CN102687027B - For the controlled tunnel gap equipment of the polymer that checks order - Google Patents

Abstract

Present invention includes composition, equipment and method for analyzing polymers and/or polymer unit. Described polymer can be homotype-or special-shaped polymer, for example, and DNA, RNA, polysaccharide or peptide. Described equipment comprises electrode, and described electrode forms the tunnel breach that polymer can pass. Electrode carrys out functionalization with reagent attached to it, and described reagent can form of short duration key with polymer unit. When form of short duration key between reagent and unit time, produce detectable signal, for analyzing polymers.

Description

For the controlled tunnel gap equipment of the polymer that checks order

To quoting of related application

The application requires U.S. Provisional Patent Application numbering in the August, 61/300,678,2010 of submitting on February 2nd, 2010The priority of the U.S. Provisional Patent Application numbering 61/378,838 of submitting to for 31st, the two is all by completely quoting and be incorporated inHerein.

Government's rights and interests

The present invention is appropriation HG004378 and the R21HG004770 being authorized by National Institutes of Health, from the national mankindThe appropriation HG004378 of the sequencing technologies plan of Joint Genome Institute, and from the appropriation of national cancer associationGovernment's support of U54CA143682 has been done. Government enjoys certain rights and interests in the present invention.

Background of invention

The new method of DNA sequencing need to reduce costs and improve availability (M.Zwolak, the M. of individualized genomicsDiVentra, ReviewsofModernPhysics80,141(2008)). In addition, longly read continuously result and will helpHelp and disclose genomic long-range structure (E.Pennish, Science318,1842(2007); A.J.Sharp, etAl., Annu.Rev.GenomicHum.Genet.ARI, 407(2006). With Sanger order-checking and method phase of future generationRatio, nano-pore order-checking (nanoporesequencing) (D.Brantonetal., NatureBiotechnology26,1146(2008)) be the technology without enzyme, utilize therein electrophoresis to force DNA molecular to pass through pore, thus sequence reading machineSystem can maintain its accuracy in the total length of molecule. Gas current through hole is responsive for the sequence in nano-pore（M.Akeson,etal.,BiophysJ.77,3227（1999）；A.Meller,etal.,Proc.Natl.Acad.Sci.（USA）97,1079（2000）；N.Ashkenasy,etal.,Angew.Chem.Int.Ed.44,1401(2005)) but all bases in nano-pore passage have been contributed current blocking (A.Meller, etal., Phys.Rev.Lett.86,3435(2001)) and in the territory, surging place that exceeds hole those (A.Aksimentiev,etal.,BiophysicalJournal87,2086（Sep,2004）；M.Muthukumar,etal.,Proc.Natl.Acad.Sci.(USA) 103,5273(2006)). As a result, using gas current to read does not still obtain single base and dividesDistinguish. Lee and Thundat propose, and the electron tunneling of crossing over DNA molecular may be enough to localize, single with sensing and qualificationNucleotides (J.W.Lee, andT.Thundat.USPatent6,905,586(2005)), this guess by Zwolak and(M.Zwolak, M.DiVentra, NanoLett.5,421(2005) supported in the calculating of DiVentra). Enter oneStep calculating show, in gap the warm-up movement of molecule widened tunnel current distribution (J.Lagerqvist, etal.,BiophysJ.93,2384（2007）；R.Zikicetal.,Phys.Rev.E74,0119191（2006）），Reduce in fact selective. In tunnel gap, the orientation range of molecule can read electrode by using chemical bond to be fastenedCome up to reduce widely (X.D.Cuietal., Science294,571(2001)), but using strong bond is not DNAThe option of order-checking must slide into next nucleotides from a nucleotides rapidly with contacting of electrode in DNA sequencing.Ohshiro and Umezawa have represented hydrogen bond can be for providing the chemistry in PSTM image to set off by contrast (T.Ohshiro, Y.Umezawa, Proc.Nat.Acad.Sci.103,10(2006)) show that these weak keys are passableServe as " sliding-contact " to individual molecule.

In application, WO2008124706A2(" checks order by identification "), 61/037647(is used for the nanotubes of DNA sequencingNano-pore "), 61/083,001(" for the series connection reader of DNA sequencing ") 61/083,993(is " for the base of the polymer that checks orderIn the equipment of CNT "), 61/103,019(" for the trans base tunnel readers checking order "), all these are by drawingBe used for merging, described the scheme of the target base contact electrode making in DNA in tunnel gap, described electrode is with being designedWith specifically with the reagent functionalization of a kind of base or another kind of base hydrogen bonding. As a result, every kind of DNA base needs differenceReader, thereby sequence must be assembled by the output of arranging four independent readers. In addition, to being designed to target spyThe dependence of the reagent of anchor point, means when two kinds of different sites are during by target (each electrode one), and electrode is essential by solelyOn the spot functionalization, this is difficult to realize in the gap of nanoscale.

Summary of the invention

The invention provides composition, equipment and method for analyzing polymers and/or polymer unit. Described polymerizationThing can be homotype-or special-shaped polymer, for example, and DNA, RNA, polysaccharide or peptide. Described equipment has electrode, and described electrode formsThe tunnel gap that polymer can pass. Electrode carrys out functionalization with reagent attached to it, described reagent can with polymerUnit forms instantaneous key. When form instantaneous key between reagent and unit time, produce detectable signal, for analyzing polymerizationThing. Configuration and regulate tunnel gap width, optimizes the signal that produces while forming instantaneous key in the unit of electrode and polymerSelectively.

Brief description of the drawings

Accompanying drawing 1 has illustrated the electrode pair T(accompanying drawing 1A by 4-sulfydryl benzamide functionalization), G(accompanying drawing 1B), C(accompanying drawing1C) and A(accompanying drawing 1D) hydrogen bonding.

Accompanying drawing 2 provides background tunnel signal exemplary in the TCB of 0.5V bias voltage. Accompanying drawing 2A is in the electric current of 10pAUnder, under the electric current of accompanying drawing 2B in 2pA.

Accompanying drawing 3 has shown the exemplary effect of electrode function for the current peak wavelength-division cloth of purine. Bare electrode (accompanying drawing3A-dA and accompanying drawing 3C-dG) obtain broad distribution (TCB intermediate gap electrical conductivity 20pS, 0.7 μ MdA, 2.9 μ MdG). ?In the log of electric current, matching is Gaussian type (referring to accompanying drawing 18-20). When an electrode distributes during by 4-sulfydryl benzene functionalizationConstriction ten times of (accompanying drawing 3B-dA, accompanying drawing 3D-dG) (gap electrical conductivity 12pS, I_bl=6pA, V=0.5V). At electric currentIn log, matching is two Gaussian types, and a peak is at i₀(" 1 ") is located, and second at 2i₀(" 2 ") locate (referring to formula III). RightIn dA, i₀=5.9pA is 5.6pA for dG. In the time that two electrodes are all functionalized (accompanying drawing 3E-dA, 3G-dG), peak electricityStream be visibly different (for dG, i₀=9.4pA, for dA, i₀=16.5pA). Accompanying drawing 3F has shown the mixture of dA and dGDistribution. The distribution at the higher peak of dA has been confirmed (accompanying drawing 3H) by the distribution at the dA measurement of concetration reducing. Accompanying drawing 3FConsistent with high electric current afterbody in 3H with the smallest number of two kinds of molecules (dA+dG) reading result. Peak width be distributed in accompanying drawingIn 29, provide.

Accompanying drawing 4 provides the electric current vs. of V=0.5V, background current=6pA in the time that adenosine is diffused in gapThe exemplary curve of time locus. Illustration has shown heaving of binding signal. All four kinds of nucleosides have been observed similar typeSignal. Referring to accompanying drawing 15.

Accompanying drawing 5 has shown the exemplary effect of the electrode function reading for pyrimidine. For reading with bare electrode (Wide distribution in accompanying drawing 5A and 5B), Gbl brings up to 40pS and improves counting rate. Narrow distribution in 5A and 5B is with being functionalizedTwo kinds of electrodes gather, dT produced to i₀=6.7pA is 13.3pA(G to dC_bl=12pS，I_bl=6pA，V=0.5V). In the solution mixing, (accompanying drawing 5C) dT peak is in the appearance of 8pA place, and dC peak occurs at 13.4pA place, has by useDistribution (Figure 21) is verified in the mixture measurement of one half strength dT.

Accompanying drawing 6 shown the current peak of two electrodes in the time being functionalized exemplary distribution (dG, V=0.5V, electric current=6pA). For the fraction of the reading result of the peak current twice of main peaks signal is synchronized with catching of two molecules in tunnel gapObtain.

Accompanying drawing 7 provides the general introduction of exemplary reading result. Accompanying drawing 7A has shown as the function of baseline electrical conductivityDA(filled squares) and dT(solid circles) peak current (under V=0.5V). Open squares (dA) and hollow circle (dT)Show that how the part of two molecule reading results is along with tunnel gap becomes less and improves. Accompanying drawing 7A has shown measurementMolecular conductivity is with G_bl(error line is ± HWHH for the circular dT of black, black squares dA) improves linearly. Two molecules readThe quantity of result (hollow circle, dT, open squares, dA) is at G_blUnder=20pS, improve, read rate is at G_blUnder=4pSEssence Shangdi has reduced. Accompanying drawing 7B provides the peak current (crosshatch of measuring in three independent operatings for four kinds of nucleosidesPost). Accompanying drawing 7B has shown in the time that two electrodes are all functionalized, has observed narrow under the characteristic electric current of every kind of nucleosidesCurrent peak distributes. Cross-hatched frame (data sets of 3 repetitions) has shown under the base current of 6pA, V=0.5V to be measuredThe peak current of every kind of nucleosides. The complete width of the CURRENT DISTRIBUTION that the error line representative of each frame top is measured. The frame of shade is aobviousShow in two electrodes an only electric current of measuring while being functionalized. The surface of functionalization and exposed Pt(light color shadePost) and the post of the dark-coloured shade of exposed Au() reading result of probe is relative insensitivity to the identity of nucleosides, as at accompanying drawing 7CIn show quantitatively, wherein relatively marked and drawed connection resistance with the molecule resistance of the probe assay of two functionalization.

Accompanying drawing 8 has shown along with extended (, tunnel current baseline, the G of exemplary tunnel gap_blBecome less), readGetting frequency reduces.

Described exemplary embodiment of the present invention accompanying drawing 9 figures.

Accompanying drawing 10A and 10B provide and have utilized gold or titanium nitride probe and gold or titanium nitride coated nano-poreExemplary tunnel gap and the details of nanohole array. Engineer's scale (110) in the electron micrograph of accompanying drawing 10B is 2nm。

Accompanying drawing 10C and 10D provide the exemplary tunnel gap of utilizing carbon nanotube probes and grapheme nano-poreDetails with nanohole array. Engineer's scale (210) in the electron micrograph of accompanying drawing 10D is 10nm.

Accompanying drawing 10E(viewgraph of cross-section) and 10F(top view) provide and utilized metal probe and carbon nanotube hole battle arrayExemplary tunnel gap and the nanohole array of row.

Accompanying drawing 11 has illustrated the gap chemical action of embodiments of the present invention.

Accompanying drawing 12 has illustrated that the gap utilizing in CNT forms the exemplary embodiment of electrode pair.

Accompanying drawing 13 has shown various amino acid whose exemplary hydrogen bonding sites.

Accompanying drawing 14 provides the exemplary chemical constitution of the nucleosides of modifying with TBDMS.

Accompanying drawing 15 provides in the TCB of two electrodes that all uses 4-mercaptobenzoic acid functionalization, 4.3 μ MdT(accompanying drawings15A), 2.9 μ MdG(accompanying drawing 15B) and 0.8 μ MdC(accompanying drawing 15C) G_blExemplary electric current-time rail of=12pSMark. The current ratio chi of accompanying drawing 15A and accompanying drawing 15B is identical.

Accompanying drawing 16 provides and has used for obtaining the gain setting of spike data, exemplary noise spectrum in open loopNoise spectrum (accompanying drawing 16B) under (accompanying drawing 16A) and SERVO CONTROL. Dash line is the matching to 1/f spectrum.

Accompanying drawing 17 has shown that fixing (5pA) of use blocks (circle), 1.5 variable σ block (square) and 2 variable σBlock that (triangle) analyze, two Gaussian logarithms and exemplary data set matching. The peak of matching moves to from 6.67.1pA is insignificant variations with respect to the separation at the peak of different nucleosides.

Accompanying drawing 18 has shown dA(accompanying drawing 18A), dC(accompanying drawing 18B), dG(accompanying drawing 18C) and dT(accompanying drawing 18D) at G_bl=20pS(V=0.5V) the exemplary CURRENT DISTRIBUTION that obtains of lower bare electrode. Under the nucleosides concentration that table 1 is listed, in 180s, recordTale is listed in each picture. Referring to embodiment 1.4.

Accompanying drawing 19 has shown dA(accompanying drawing 19A), dC(accompanying drawing 19B), dG(accompanying drawing 19C) and dT(accompanying drawing 19D) at G_bl=40pS(V=0.5V) the exemplary CURRENT DISTRIBUTION that obtains of lower bare electrode. Notice, due to the width distributing, can obtainSome of dA are reading result clearly. With at G_blThe data that obtain under=20pS are compared, the read rate between purine and pyrimidineLess different. Referring to accompanying drawing 18. Counting in the 180s cycle is listed on each picture.

Accompanying drawing 20 has shown to be marked and drawed on double-log plot, to the exemplary parabola of the data from accompanying drawing 19AMatching (solid line).

Accompanying drawing 21 provides the exemplary distribution of the mixing of dT and dC, and what the relative concentration of dT used in accompanying drawing 5C is to subtractHalf. Distribute and three Gaussian logarithmic functions (solid line) matching. DT peak position is in 6.6pA, and dC peak position is in 12.3pA. HighElectric current afterbody and fraction dG+dC reading result matching (concentrating on 6.6+12.3pA).

Accompanying drawing 22A and 22B provide with the 0.75V bias voltage (G that produces larger gap_bl=8pS) at I_blUnder=6pAThe exemplary CURRENT DISTRIBUTION of measuring. The reduction of the electrical conductivity according to expectation of molecule (G(dA)=14.4pS, G(dC)=15.6pS). Be greater than from the reduction of dA that the matching that shows accompanying drawing 7A estimates. This shows except the index at gap lengthOutside dependence, there is bias-dependent. The data (referring to accompanying drawing 23) that gather as the function of bias voltage under the size of fixed interval (FI)Tend to confirm this trend.

Accompanying drawing 23 provides the exemplary peak current (12pS as the dA of the function of bias voltage under constant clearance electrical conductivity;0.75V, 9pA, 0.5V, 6pA and 0.25V3pA). Circle is the data that gather with an exposed gold electrode, theyShow the slight dependence to bias voltage. Show along with bias voltage falls by the data (square) that the electrode of 2 functionalization gathersLow peak current improves. The sign that changes bias voltage does not cause large change (data under 0.5V).

Accompanying drawing 24 has shown as the reading result function of baseline tunnel current, that functionalize tip is per second, has been to pass throughBy between 180s to dA(circle) and dT(square) the data equalization that obtains obtains. These data depend on a littleProbe geometry, this is in a kind of error line being reflected in by relatively obtaining by the data of two different probe collectionsEffect.

Accompanying drawing 25 has shown for dA at G_blThe exemplary CURRENT DISTRIBUTION (probe of functionalization) obtaining under=20pS,2 and the evidence of 3 molecule reading results are even shown.

Accompanying drawing 26 has shown I_blUnder=6pA, V=0.5V to dT(circle), dG(square), dC(triangle) and dAThe distribution of the exemplary matching of the experimental data collection of (rhombus) (comprise 2 molecules read peaks) (peak from left to right: dT, dG,DC, dA). Locate to arrange level of resolution in shown value (8pS, 11.7pS and 14.8pS), if dT i < 8pA is produced72% probability, be 64%(8pA < i < 11.7pA to dG), be 61%((11.7 < i < 14.8pA to dC), and be 60% to A（i>14.8pA）。

Accompanying drawing 27 has shown the spike (accompanying drawing 27A) that retains all records in the analysis by exemplary, and passes through((40 μ s) exemplary electric current that the spike (accompanying drawing 27B) of sampled point perdurabgility obtains divide 20 s) or 2 of μ to eliminate only 1Cloth. Data are for dG, 2.9 μ M, G_bl=12pS. Solid-line curve be with A in peak independently, and with B at i₀And 2i₀Locate the 2Gaussian logarithm matching at fixing peak. Data in A are leading by the feature at 7.3pA place, equal by functionalization notThe electric current of probe records. Peak in B moves to 9.7pA. Distribution has reflected the limited frequency sound of STM current-to-voltage converterShould, it will reduce the amplitude that records at peak (referring to accompanying drawing 29) fast. To dC obtained similar result (peak of all data=7.3pA, the peak=13pA of the data of filtration). (peak=7.3pA of all data filters in the impact that the data of dT are not filteredThe peak=6.8pA of data).

Accompanying drawing 28 provides exemplary CURRENT DISTRIBUTION, and it is by retain all records in the exemplary analysis of dASpike obtain (all data of accompanying drawing 28A-, accompanying drawing 28B-eliminates two the fastest points). In this case, 6.5pA placeThe residue at " electrode of individual feature " peak retains, and now 1 or 2 some spikes are eliminated, thus data and threeThe matching of Gaussian logarithmic function, has two independently peak value (i₀，i₁And 2i₁). In the time that the shortest spike is eliminated, distributeMaximum move to 15.6pA from 6.5pA. The shape distributing depends on the probe of use, and 2Gaussian matching is to otherData set work good (for example, accompanying drawing 2), wherein " not functionalization " peak is almost fully disappeared by getting rid of the fastest spikeRemove.

Accompanying drawing 29 has shown dA(accompanying drawing 29A), dC(accompanying drawing 29B), dG(accompanying drawing 29C) and dT(accompanying drawing 29D) the spike longevityThe exemplary distribution of life. Round wire is bare electrode, and triangle line is the electrode of a functionalization, and square line is twoThe electrode of functionalization. Sharp-pointed feature has reflected data binning. The working concentration that all data show at table 1 and 0.5VLower collection. For exposed probe G_bl=20pS, the probe of functionalization is 12pS. 40 or the 20 μ s duration of arrow pointsSpike, they are eliminated to strengthen selectively from CURRENT DISTRIBUTION. Except dT, with the probe life-span of two functionalization beSlightly long. But, the life-span of exposed probe, or the life-span of a functionalize tip be substantially the same. Thereby tunnel distributesNarrow must be reflected as functionalization and naked metal surface between difference in the combination range of geometries of allowing, instead ofIn conjunction with the difference in the state life-span. Current-to-voltage converter-3dB frequency is ~ 7kHz(143 μ s), thereby show hereinIn data, feature is weakened faster.

Accompanying drawing 30 has illustrated exposed gold thread exemplary in the 50mM potassium ferricyanide (electromotive force vs.Ag line).

Accompanying drawing 31 has illustrated the cyclic voltammetry at the coated STM tip of exemplary HDPE. Suppose the point that hemispherical exposesEnd shape and the formula of use I_max=2 π RnFCD, the exposed surface area of coated scan-probe is 10^-2μm²The order of magnitude on.

Accompanying drawing 32 has illustrated the exemplary of 4-sulfydryl benzamide individual layer (line of below) and powder (line of top)FTIR spectrum.

Accompanying drawing 33 is STM images, has shown the island of sulfydryl benzamide on Au surface. , 0.5V most advanced and sophisticated at gold is most advanced and sophisticated partiallyPress the image in the 1mMBP buffer solution of 10pA set-point.

Accompanying drawing 34 has shown from the optics of exemplary electrodes and transmission electron microscope (TEM) image. Accompanying drawing 34A is nakedThe optical imagery of electrode. Accompanying drawing 34B and 34C are the TEM images of bare electrode. Accompanying drawing 34D is the optical imagery of coated electrode. AttachedFigure 34 E and 34F are the TEM images of coated electrode. Dash line arc in 34C has the radius of 16nm. Arrow in 34E and 34FHead shows the golden position exposing.

Accompanying drawing 35 has illustrated and has used bare electrode probe and the thump telegraph repeater of functionalized electrode surface in water. When probe and tableWhen face is all exposed, and in PB when surface and/or probe are while being exposed, see similar signal.

Accompanying drawing 36 has shown pure H₂O(marks and draws many curves in each case) in, exposed gold electrode (accompanying drawing 36A),The tunnel current decay of the electrode (accompanying drawing 36B) of functionalization and an electrode (accompanying drawing 36C) exposed and a functionalizationCurve.

Accompanying drawing 37 has shown the exemplary histogram of β, the negative value of logarithmic decrement slope of a curve, that is,。In pure water to exposed gold electrode (accompanying drawing 37A), with the electrode (accompanying drawing 37B) of sulfydryl benzamide functionalization and one exposedWith an electrode by sulfydryl benzamide functionalization (accompanying drawing 37C) obtain value. Gausian matching (mean value ± SD) is producedRaw: 37A → 6.11 ± 0.68nm^-1,37B→14.16±3.20nm^-1; And 37C → 6.84 ± 0.92nm^-1。

Accompanying drawing 38 shown utilize 4-sulfydryl benzamide reader (reader) molecule gather d(CCACC) demonstrationThe 10s time locus of property. Note the advantage of a-signal. Current spike distribution (illustration) is almost dominated by a-signal completely, in matchingC part (black line) be 7% or lower. This has shown that probe spends the A base that the more time is attached to minority.

Accompanying drawing 39 has shown for dAMP(accompanying drawing 39A), dCMP(accompanying drawing 39B), dGMP(accompanying drawing 39C) and dmCMP(accompanying drawing39D), along with the exemplary current spike track in past of time, show the exemplary outburst of data. These examplesEach by the galvanic areas without spike around.

Accompanying drawing 40 illustrated cytidine (grey) and^5meCytidine uses benzoic acid reader to measure in trichloro-benzenes solventExemplary CURRENT DISTRIBUTION.

Accompanying drawing 41 has shown dGMP, dCMP, dAPM and d^mThe exemplary distribution of " the on time " of CMP monomer, solid line refers toThe matching of number property.

Accompanying drawing 42 has shown d(C)₅、d（A）₅And d(^mC）₅The exemplary distribution of " the on time " of polymer, solid line refers toThe matching of number property.

Accompanying drawing 43 has shown dGMP, dCMP, dAPM and d^mThe exemplary distribution of " the off time " of CMP monomer, solid line refers toThe matching of number property.

Accompanying drawing 44 has shown d(C)₅、d（A）₅And d(^mC）₅The exemplary distribution of " the off time " of polymer, solid line isIndex matching.

Accompanying drawing 45 has shown d(A)₅Spike > the exemplary distribution of 0.1nA counting. These are total approximately 20%,DNTP or d(C)₅In do not observe.

Accompanying drawing 46 has shown d(^mC）₅Spike > the exemplary distribution of 0.1nA counting. These are total approximately 20%,DNTP or d(C)₅In do not observe.

Accompanying drawing 47 has shown and benzamide surface (R: the 2-deoxyribosyl-5-sodium ascorbyl phosphate that does not contain DNA base) phaseThe exemplary spr sensor figure of nucleosides-the 5 '-monophosphate (A, C, G, T, R) of mutual effect. Lines are curves of matching, are builtMould is described 1:1 binding events.

Accompanying drawing 48 has shown the histogram that adheres to event, is a pair of 4-sulfydryl benzamide reader of trapping dAMP is dividedSon records with AFM. Accompanying drawing 48A has shown the control curve gathering while not there is not dAPM, and it shows benzene hardlyBetween formamide molecule, adhere to event, supposition is because they are broken by water resistance. Accompanying drawing 48B has shown after cleaning for the first timeDAMP adheres to. Accompanying drawing 48C has shown the adhering to of dAMP after cleaning for the second time. Accompanying drawing 48D has shown in cleaning for the third timeAdhering to of dAMP afterwards. Accompanying drawing 48E has shown the adhering to of dAMP after the 4th time is cleaned. Add dAMP and cause many things of adhering toPart, it,, along with excessive dAMP is washed out system and improves (accompanying drawing 48B, C), reduces along with cleaning continuation (accompanying drawing 48D, E).

Accompanying drawing 49 has shown that displacement (top pattern-black) and the electric current (bottom pattern-grey) of exemplary simulation is rightThe more m-ladder than time, wherein correlation C has 0.9 value.

Accompanying drawing 50 has shown that displacement (top pattern-black) and the electric current (bottom pattern-grey) of exemplary simulation is rightThe more m-ladder than time, wherein correlation C has 0.98 value.

Accompanying drawing 51 has shown that displacement (top pattern-black) and the electric current (bottom pattern-grey) of exemplary simulation is rightThe more m-ladder than time, wherein correlation C has 0.99 value.

Accompanying drawing 52 has shown the exemplary standardized distribution of the signal obtaining from homopolymer. It is right that accompanying drawing 52A has shownThe matching of standardization CURRENT DISTRIBUTION. Accompanying drawing 52B has shown at the peak frequency of signal outburst Plays, has been and fitting of a polynomial. To the matching distributing be used to specify specific noise outburst from the probability of A or C (if average current and frequency are being intersectedAbove or below point, be labeled as " IAC " and " fAC "). C and^mThe CURRENT DISTRIBUTION of C be separate (crosspoint is labeled as " I_mC"), butThat frequency distribution is overlapping.

Accompanying drawing 53 has shown the exemplary hydrogen of 4-sulfydryl benzamide to adenine, thymidine, cytimidine and guanineKey bonding mode.

Accompanying drawing 54 has shown the exemplary data that read single base in heteropolymer. 54B has shown exemplary tunnelNoise outburst, large, rare spiking is C, less, spiking is A more frequently. The spike right and wrong of " * " markSpecific. Accompanying drawing 54c has shown the rolling mean value (0.25s window, 0.125s ladder) of spike height. C base has producedLower than 0.015nA(straight line) insignificant spike number. Accompanying drawing 54D has shown the rolling mean value of peak frequency. Accompanying drawing 54EShown that signal is shown by light gray line from A() or C(shown by dark-coloured line) probability.

Accompanying drawing 55 has shown the tunnel signal of the tunnel gap of functionalization in phosphate buffered saline (PBS), but does not have analyzedThing, uses 20pS gap (i=10pA, V=+0.5V). Except the lines noise of pointed some AC coupling of arrow itOutward, this embodiment obtains undistinguishable signal.

Accompanying drawing 56 has shown the tunnel signal of the tunnel gap of functionalization. Accompanying drawing 56C-F shown when nucleotides dAMP(attachedFigure 56 C), dCMP(accompanying drawing 56D), d^mCMP(accompanying drawing 56E) and dGMP(accompanying drawing 56F) the characteristic electric current point that produces while being introduced intoPeak (dTMP does not obtain signal in this embodiment). Accompanying drawing 56G-J has shown dAMP(accompanying drawing 56G), dCMP(accompanying drawing56H）、d^mCMP(accompanying drawing 56I) and dGMP(accompanying drawing 56J) the corresponding distribution of pulse height. Curve in accompanying drawing 56G-JTwo Gaussian in electric current logarithm distribute.

Accompanying drawing 57 has illustrated the parameter for characterizing tunnel signal. If exceeded noise standard deviation in local background1.5 times, spike is counted. There is (duration T B, frequency f B) in signal, contain separately the electricity under frequency f S in outburstStream spike. For cycle t_onSpike is high, for cycle t_offLow. Gross-count rate (referring to accompanying drawing 56G-J) is all quick-friedSpike number in sending out is divided by the measured value of time.

Accompanying drawing 58 has illustrated distributing from the tunnel signal of oligomer is how to be similar to those that form nucleotides. Accompanying drawing58A, C and E have shown d(A)₅、d（C）₅And d(^mC）₅Representational current locus, be distributed in accordingly in accompanying drawing 58B, D and FShow. Curve in accompanying drawing 58B, D and F has shown the matching to constitutive character nucleotides and oligomer nucleotides. Curve thatThis is closely relevant. Accompanying drawing 58G and I have shown from the oligomer d(ACACA mixing) (accompanying drawing 58G) and d(C^mCC^mCC) (attachedFigure 58 I) current locus, and corresponding CURRENT DISTRIBUTION (accompanying drawing 58H and J). Block curve in accompanying drawing 58H and J has been scalarHomopolymer matching. In accompanying drawing 58H, top chain-dotted line has shown A contribution, and the chain-dotted line of bottom has shown C contribution. At accompanying drawingIn 58J, top curve has shown^mC contribution, bottom curve (0.02 place starts in electric current (nA) axle top) has shown C contribution. NumberAccording to being described well by the homopolymer parameter (being labeled as " 1 ") through some M signal, new high current characteristic (is labeled as" 2 ") show that sequence background affects and reads slightly. Top horizontal line mark in accompanying drawing 58G C sample signal, and the horizontal stroke of belowWire tag A sample signal. Top horizontal line mark in accompanying drawing 58I C sample signal, the horizontal line mark of below^mC sample signal.

Accompanying drawing 59 has shown about the data (life-span is on the order of magnitude of second under zero-g) in life-span that read compound. AttachedFigure 59 A has shown AFM gap function, and wherein s shape lines have represented 34nmPEG joint. Accompanying drawing 59B has shown representationalForce curve, has shown (i) once to pull to exceed a molecule, and power baseline does not recover after each fracture, and z-extends (correctionMost advanced and sophisticated displacement) be ~ 34nm, and the unimolecule curve of the type that (ii) exemplary software is accepted (as A.Fuhrmann,PhDThesisinPhysics, ArizonaStateUniversity, 2010 is described). Power is after bond fissionGet back to baseline, the extension of correction is ~ 34nm. Accompanying drawing 59C has shown the histogram that pulls bond fission power under speed at mark.Curve has shown the exemplary maximum likelihood matching with allos key model. Accompanying drawing 59D has shown contrast key model parameter markThe key survival probability (solid line) of painting, it is 5000nm/s, 2000nm/s, 500nm/s and 200nm/s from top to bottom. TheseMatching has produced zero-g off and has led 0.28s^-1, mean that the assembly time-to-live is on the order of magnitude of second, than molten in nano gapLife-span in liquid more of a specified duration many (solution in conjunction with the details of measuring referring to accompanying drawing 47).

Accompanying drawing 60 has illustrated chip according to the embodiment of the present invention. In chip, a pair of electrode that reads passes through conductorThe ald of for example TiN forms, and (for example, 2nm) dielectric layer that described conductor dbus is excessively thin is separated, and has and bores chipNano-pore. Identification molecule (reagent) covalency is tethered on metal electrode, along with electrophoresis drives molecule by gap, passes through subsequentlyNoncovalent interaction is formed on the connection of the upper self assembly of each base (or residue).

Accompanying drawing 61 has shown the exemplary hydrogen of imidazoles-2-carbamyl to adenine, thymidine, cytimidine and guanineKey bonding mode.

Accompanying drawing 62A and 62B shown in the fixed interval (FI) that comprises imidazoles-2-carbamyl functionalized electrode with deoxidation-The exemplary CURRENT DISTRIBUTION that nucleotides is measured.

Accompanying drawing 63 has shown have the electrode of imidazoles-2-carbamyl functionalization and can constant clearance put down from the teeth outwardsThe demonstration equipment of the probe moving.

Accompanying drawing 64 has shown DNA reading result matching dC₅(accompanying drawing 64A) and dA₅The nucleotides CURRENT DISTRIBUTION of (accompanying drawing 64B).

Accompanying drawing 65 has shown the exemplary electric current reading result of the AAAAA oligomer of fixed interval (FI).

Accompanying drawing 66 has shown the exemplary electric current reading result of the CCCCC oligomer of fixed interval (FI).

Accompanying drawing 67 has shown the d(of fixed interval (FI)^mC）₅The exemplary electric current reading result of oligomer.

Accompanying drawing 68 has shown the d that remains on the variable gap under approximately constant value by the SERVO CONTROL of tunnel current(CCCCC) the exemplary electric current reading result of oligomer.

Accompanying drawing 69 has shown dA₅(slope of top) and dC₅The signal explosion time contrast scans speed on (slope of below)Exemplary plot reciprocal, the about 0.3nm of slope.

Accompanying drawing 70 has shown the exemplary electric current reading result of the ACACA oligomer of fixed interval (FI).

Accompanying drawing 71 has shown the exemplary electric current reading result of the CCACC oligomer of fixed interval (FI).

Accompanying drawing 72 has shown the C of fixed interval (FI)^mCC^mThe exemplary electric current reading result of CC oligomer.

Accompanying drawing 73 has shown the ACACA that remains on the variable gap under approximately constant value by the SERVO CONTROL of tunnel currentThe exemplary electric current reading result of oligomer.

Accompanying drawing 74 has shown by the SERVO CONTROL of tunnel current and has remained on variable gap under approximately constant valueC^mCC^mThe exemplary electric current reading result of CC oligomer.

Accompanying drawing 75 has shown by the SERVO CONTROL of tunnel current and has remained on variable gap under approximately constant valueThe exemplary electric current reading result of GTCGTCGTC oligomer.

Accompanying drawing 76 has shown the exemplary identification molecule (reagent) of amino acid and peptide trunk.

Detailed description of the invention

The invention provides composition, composition, equipment and method for analyzing polymers unit and/or polymer. CanComprise heteropolymer and relevant unit with analyzed exemplary polymer unit and polymer. For example, can be analyzedPolymer comprises DNA, RNA, polysaccharide and peptide; Polymer unit comprises polymer monomer, nucleotides, nucleosides, amino acid, polysaccharide listBody. In some embodiments, can analyze the mark outside heredity, for example methylated DNA and/or RNA, and with for example non-firstThe DNA/RNA unit of base is distinguished mutually.

Described equipment comprises with two of one or more reader molecule (at this also referred to as reagent) functionalization or moreMultiple electrodes, and the tunnel gap that can pass of polymer unit and/or polymer. Reagent on electrode can with polymerUnit form instantaneous key. In the time that unit is in gap, form instantaneous key chemical or physics, and complete firstAnd second loop between electrode. Then the instantaneous key forming can cause the detectable signal for analyzing polymers.

Electrode

Two or more electrodes can be made up of any applicable material, and described material can be used can combining target polymerizationThe reagent of thing unit carrys out functionalization. For example, electrode can be made up of the material of any conduction, for example, metal, metal alloy, gold,Platinum, billon, platinum alloy, carbon, CNT, Graphene or titanium nitride. In some embodiments, electrode comprise probe andSubstrate. Electrode can be on any applicable inorganic or organic insulation or between form or by its SI semi-insulation, exampleAs, inorganic material comprises Si_xO_-1x, silicon nitride, metal oxide, or organic material comprises polymer, for example polyethylene, polyphenylEthene, polymethyl methacrylate and well known in the art other. Insulating materials can be configured to stop and flow at electric currentThe ambient noise of self-electrode when moving. For example, except little tip or top, electrode can fully cover with HDPE.In another embodiment, electrode can be embedded between insulating barrier, only exposes the region (accompanying drawing 60) contacting with nano-pore.Nearly a square micron can be exposed to the salting liquid that reaches a mole, only has insignificant for half volt or lower bias voltageLeakage current.

Reading reagent plays an important role in " sharpening " electrode. Typical gold electrode has the crystal composition of receiving, Qi Zhong greatLittle 10nm or larger contact-making surface are exposed. Thereby to seem impossible be only to contact single base. But, when electrode quiltWhen functionalization, single base is easily to differentiate. This is because tunnel electrode has been served as in the contact of specific molecule now, at goldOn metal surface, form sharp-pointed, well-defined aculea.

Reagent

One or more of reagent functionalization for electrode. Electrode can carry out functionalization with the combination of same reagent, reagent, orBy the functionalization respectively of reagent independently. Can use can be in conjunction with electrode combining target polymer unit any suitable instantaneouslyThe reagent closing.

In order to promote the combination of subject polymer unit and electrode, various functional groups can be tethered to energy and target polymerizationOn the reader molecule of thing unit combination, depend on the electrode substance of expectation. Applicable functional group can comprise, for example ,-SH、-NH₂、-N₃、-NHNH₂、-ONH₂,-COOH ,-CHO, acetylene, dithiocar-bamate and dithionate. Pass throughMolecular entergy level is aimed at the fermi level of metal more closely, connected key with the dithiocar-bamate of metal and improve widelyTunnel current (referring to FlorianvonWrochem, DeqingGao, FrankScholz, Heinz-GeorgNothofer,GabrieleNellesandJurinaM.Wessels,“Efficientelectroniccouplingandimprovedstabilitywithdithiocarbamate-basedmolecularJunctions ", NatureNanotechnology, June20,2010). In some embodiments, electrode is fastenedTo functional group, functional group is then in conjunction with reader molecule. For example, for metal, in the time that electrode is gold, there is mercaptan meritThe reagent of energy group can be for facilitating the covalent bond between reagent and electrode. Dithiocar-bamate can be for being attached toGold, Pt and TiN. These groups can provide the electronics coupled of the enhancing between metal and reagent. Amine chemical action can be forFunctionalization graphene hole and CNT end, because Graphene edge has carboxylate, carbonyl and epoxides frequently.

Reagent can with subject polymer unit (J.Heetal., Nanotechnology20,075102(2009)) and nucleosides (S.Changetal., NatureNanotechnology4,297(2009)) form instantaneousKey. Instantaneous key can be key physics, chemistry or ion, as long as described key allows to go out to detect electricity by electrode detectionSubsignal (S.Changetal., Nanotechnology20,075102(2009); M.H.Lee, O.F.Sankey, Phys.Rev.E79,0519111(2009)). Preferred instantaneous key comprises hydrogen bond. Thereby, exemplaryReagent can comprise hydrogen supply or accept group. Another embodiment is the stacking interaction of the pi-between aromatic rings, fragranceRing is pulled to together in water or aqueous electrolyte.

Comprise mercaptobenzoic acid, 4-sulfydryl benzamide, imidazoles-2 – carbonyl in conjunction with the exemplary reagent of DNA and/or RNACompound and aminodithioformic acid imidazoles-2 – carbonyl compound are (also referred to as 4-carbamyl phenyl aminodithioformic acidSalt). 4-sulfydryl benzamide has presented two hydrogen bonds and has supplied with site (on nitrogen), and a hydrogen bond acceptor site (carbonyl).In accompanying drawing 53, show the possible binding pattern to for example four kinds of nucleotide bases. In accompanying drawing 61, show imidazoles-2-ammoniaThe possible binding pattern of base formyl and four kinds of nucleotide bases. Relate to the pi-between aromatic rings and the DNA base in readerOther stacking bonding patterns are also possible. When being in various solvents, for example organic solvent, water or aqueous electrolyte solutionIn, reagent can be formulated to present one or more hydrogen bond donor and/or one or more hydrogen bond acceptor. For example,Mercaptobenzoic acid is for example worked in trichloro-benzenes at organic solvent, and 4-sulfydryl benzamide imidazoles-2-carbonyl compound and two sulfo-sCarbamic acid imidazoles-2 – carbonyl compound is worked in aqueous electrolyte and water. Be noted that and comprise these design principlesMany molecules will work as reader (reader). For example, be anchored on gold or TiN electrode with thiol functionGuanine will produce identification signal.

In some embodiments, reagent can be configured to comprise flexible part, and it forms the hydrogen of electrode and reagentBridge between key bonding part divides. This bridge can be that replace or unsubstituted alkyl chain, for example, and-(CH₂）_y-, wherein y 1 arrives5 integer. For example, in the time being functionalized on electrode, have-CH of imidazoles-2-carbamyl₂CH₂-bridge, connects amide moietiesTo electrode. This bridge allows amide moieties rotation, thereby fast with different and detectable mode and adenine, cytimidine, birdPurine and thymidine interact. Referring to accompanying drawing 61.

Reagent can also be configured to form instantaneous key with amino acid and analyze peptide. Accompanying drawing 13 has shown on amino acidThe example in hydrogen bond donor and acceptor site. Near site (mark " D " donor, or " A " acceptor) two or more isObtainable on asparagine, glutamic acid, glutamine, histidine and arginine. Lysine, serine, threonine, junket ammoniaSingle locus on acid and tryptophan can read with the reagent of peptide trunk formation hydrogen bond in conjunction with utilizing also. Aromatic agent canTo identify the amino acid (histidine, tyrosine, proline and tryptophan) with aromatic rings by the stacking mode of pi-. Thereby,Peptide can be according to describing equipment at this and method is analyzed. Accompanying drawing 76 has shown identification polypeptide trunk and has identified amino acid whose side chainExemplary reagent.

Tunnel gap

The unit of polymer, for example, the nucleosides of DNA or the amino acid of protein along with they diffuse through tunnel gap,Or by electrophoresis drive by time be detected. The width in gap can be fix or dynamically adjust. Gap preferablyFix. Gap comprises two spaces between electrode. Gap is adjusted to such size, thereby each object element is applicable toGap. Gap can be approximately 0.5 to the width of about 6nm, for example approximately 1 to approximately 4, approximately 1.5 to about 3.5nm, approximately 2 arrive about 3nm,Or approximately 2 to about 2.5nm. Gap width can depend on that the reagent of use changes with the subject polymer unit that will analyze. RightIn two electrodes by 4-sulfydryl benzamide functionalization, gap can be approximately 2 to about 2.5nm, and for example, approximately 2.1 arriveAbout 2.2nm, or about 2.16nm(is in the time that gap electrical conductivity is 20pS, is generally used for DNA and reads). For using imidazoles-2-aminoTwo electrodes of formyl functionalization, gap can be approximately 2.2 to approximately 2.6, for example, approximately 2.3 arrive about 2.5nm, approximately 2.35 arriveAbout 2.4nm, or about 2.37nm(is in the time that gap electrical conductivity is 20pS, is generally used for DNA and reads). Clearance distance can as withDetermining of lower description. Accompanying drawing 1 has shown in a fixing tunnel gap uniqueness with each formation of four kinds of DNA nucleosidesHydrogen bonding. Each of 4-sulfydryl benzene and four kinds of nucleosides forms hydrogen bond. Hydrogen bond is circular, and " S " represents deoxyriboseSugar moieties. These structures produce by computer simulation, may very well represent actual structure, because 4-sulfydryl benzene firstAcid amides uses in organic solvent. For the reader molecule of working in water, with hydrone competition hydrogen bond andThe interaction of the aromatic rings being mediated by water, makes the trial of structural modeling complicated, and aromatic rings is pushed together formation by waterThe stacking interaction of pi-. The definite size in gap may be important aspect the reliable reading result of acquisition. It should be adjustedWhole size, thus most of the time (or produce most of signal) be by gap only a polymer unit there is instituteCause. Applicable gap width can be by using the dynamically equipment of adjusting play to determine. In some enforcement sideIn formula, the equipment of dynamically adjusting can be for evaluating objects unit. In both cases, gap width can be determined as followsOr arrange: electrode is close to each other to together, until selected tunnel current arrives specific bias voltage. For example,, when at 1,2,4-tri-In chlorobenzene, when built tunnel, under 0.5V bias voltage, the electric current of 6pA is corresponding to the gap of 2.5nm. Micro-by application scanning tunnelIn mirror field, known active SERVO CONTROL maintains gap. In some embodiments, SERVO CONTROL has and is limited to 100Hz'sResponse frequency or lower. Suppose that gap is kept enough large, background tunnel signal does not have feature, as shown in Figure 2. When dividingSon in gap in conjunction with time, in tunnel gap, observe instantaneous current peak (accompanying drawing 4). In the time that two electrodes are functionalized,Under distinctive electric current, observe the narrow distribution (Fig. 7 B) of current peak for every kind of nucleosides. But, be attributable in gapExceed the target molecule combination of, the high electric current afterbody on distributing simultaneously, make to identify specific nucleosides from current signalComplicated. This can be created in second peak in CURRENT DISTRIBUTION, is the twice (accompanying drawing 6) of main peak electric current. Along with make in gapLess (baseline electrical conductivity or the electric current of raising), thus more contacts of leap electrode become possibility, polymolecular reading resultRelative frequency improves. This frequency progressively improving of two molecule reading results shows that in accompanying drawing 7A (it has also shown peakHow electric current improves along with reducing gap). Thereby, make gap there are more greatly two kinds of adverse effects: first, peak current falls(as shown in accompanying drawing 7A), the second, for given aimed concn, read rate reduces (accompanying drawing 8).

Thereby in some embodiments, important element comprises that (a) introducing has nano-pore, variable and can controlTunnel gap, can be shifted to reading system and once present an element (, by described nano-pore subject polymerFor DNA polymer, next base); And (b) use in a certain way or other modes in conjunction with the examination of all targetsAgent, gap is adjusted to such size, thereby only has the combination geometry of minority uniqueness for target, thereby produces districtOther property signal.

Nano-pore

In some embodiments, equipment can have one or more nano-pore, by described nano-pore polymerCan be directed to tunnel gap for analyzing. Nano-pore can be configured to allow polymer once to flow one to tunnel gapUnit. Thereby, can be less than the nano-pore for analyzing peptide for the nano-pore of analyzing DNA.

Connect for the tunnel with variable gap, such embodiment has been described in accompanying drawing 9. Gap itself thinJoint provides in accompanying drawing 10A-F. The details of fixing gap device provides in accompanying drawing 60, such as DNA of the polymer that check orderBe present in fluid reservoir 1 and (show with cross section). The fluid that contains polymer can flow through the array of nano-pore 3, or appointsSelection of land can be by electrophoresis bias voltage V_eDrive by nano-pore, described bias voltage by reference to electrode 4 at the first holder 1 and secondBetween body holder 2, apply. Two holders are all full of electrolyte, for example, 1MKCl, in addition, the pH value of holder 1 canBe adjusted to large value (pH=11 or 12) target dna is maintained to its single stranded form. Come solely according to the signal of telecommunication if do not neededVertical checking displacement, the electrolyte solution in collection holder 2 is preferably made little (mM) and is minimized electrochemical seepage. ?In some embodiment, nano-pore is to conduct electricity in electricity, connects by the electrode 5 that is layered on nanohole array top. ArrayCan be by surveying by scanning conductor 7 one or more second electrode 6 in position, scanning conductor 7 for example, is sweptRetouch the known x in probe microscope field, y, z scanning element. Scanner can be attached to nanohole array by rigid frame 8.

Exemplary being shown in accompanying drawing 10 that tunnel connects shows. Accompanying drawing 10A has shown that Au probe reads sequence, along withThe probe 104 and gold or TiN103 of DNA molecular on nano-pore 101 tops, DNA is shifted and is passed through nanometer by electrophoresisHole 101. Nano-pore 101 shows in cross section, and it drills silicon, silicon nitride or silica substrate 102. Reading reagent 105 Hes106 are attached to probe 104 and metal electrode 103. Electron micrograph in accompanying drawing 10B has shown the suprabasil nanometer of silicon nitrideHole 107, it is coated by the thin layer (20nm) of gold 108. The action that gets out hole causes gold recrystallization around hole, thereby golden109 projection sharp-pointed, atom level is outstanding at the edge of nano-pore, and what form polymer reads one of electrode.

Accompanying drawing 10C has shown an embodiment, is wherein maintained at Graphene with the probe of carbon nanotube electrode 204On nano-pore 201 in substrate 202, the graphene-based end 202, was supported by the nitride silicon based end 203. Reading reagent 205 and 206 adheres toTo the edge of CNT204 and grapheme nano-pore 201. Electron micrograph in accompanying drawing 10D has shown in Graphene multilayerThe nano-pore 207 getting out on 208.

Accompanying drawing 10E and 10F have shown an embodiment, are wherein maintained at from the outstanding metal probe 13 of insulator 12Protrude from the array in carbon nanotube hole of dielectric substrate 11 (nano-pore is marked as 10) described dielectric substrate 11Coated with thin metal electrode 5, CNT is outstanding by described metal electrode 5. Such array can be by nanotube from siliconSurface CVD growth, be accompanied by and etch away basic-level support thing silicon nitride and fill to manufacture subsequently, as Holtetal.,FastMassTransportThroughSub–2-NanometerCarbonNanotubes.Science,2006.312:p.1034-1037 described, be incorporated in by reference this. For example, all the other are prominent removing by ion(ic) etchingBefore the CNT going out, the end face of film can have evaporation Au layer thereon and be used as contact (thickness 10 to 100nm).DNA is shifted recently by Liuetal. by the electrophoresis of CNT, Translocationofsingle-strandedDNAthroughsingle-walledcarbonnanotubes.Science, 2010,327, p64-67 represents, be incorporated in by reference this. Probe 6 can cover with the layer of insulator 12, leaves the top that a small amount of (a few square micron) exposesEnd 13, as Nagaharaetal., PreparationandCharacterizationofSTMTipsforElectrochemicalStudies.Rev.Sci.Instrum., 1989.60:p.3128-3130 is described.This generation enters the minimum electrochemistry leakage current of electrolyte solution.

In some embodiments, parts as above can be configured in microarray or chip, as for example attachedIllustrated in Figure 60. At this, support material 302(silicon, silica or silicon nitride) coated with thin metal electrode 303, for exampleThe TiN depositing by ald, then covers with the layer of dielectric 304, and its thickness 308 is according to the examination of reading usingAgent optimal selection. For most of reagent described here, this thickness is 1.5 to 3nm. Deposit the second electrode 305, andCover with last dielectric layer 309. Nano-pore 301 is drilled passes whole equipment (by the mode of electron beam, as known in the art), reading reagent 306 and 307 functionalization for the surface of metal electrode of exposure.

Metal electrode can form by any conventional method around nano-pore. For example, Pt can be in silicon nitride filmOn the nano-pore forming, deposit by FIB chemical vapor deposition. Metal, for example TiN, also can pass through atomic layerDeposition or chemical vapor deposition deposit, etching afterwards or generation hole. First metal also can be coated on film, for example,SiN, Si or SiO₂, after this get out hole.

Graphene, a kind of material conducting electricity itself, also can be as the electrode of nano-pore. The in the situation that of Graphene, holeHole can get out in Graphene. The displacement in Graphene hole can, for long oligomer, for example, reach 48kbp. UsingWhen Graphene, the likely edge of functionalization hole only. In accompanying drawing 11, show for nano-pore and utilized showing of CNTThe tunnel gap of plasticity. Probe can be removed from substrate, thus substrate with on probe, be can by different reagent functionalizationCan. In some embodiments, probe, gold or platinum or platinum alloy carry out functionalization with 4-sulfydryl phenylurea 21, and 4-sulfydryl phenylurea isIn aqueous solution, present the reagent of hydrogen bond donor and acceptor. The end of CNT 10 preferably can utilize this area public affairsThe amido link (not shown) urea part 22 of knowing is carried out functionalization. Referring to Feldman, A.K., M.L.Steigerwald, X.Guo,andC.Nuckolls,MolecularElectronicDevicesBasedonSingle-WalledCarbonNanotubeElectrodes.Acc.Chem.Res.,2008.41:p.1731-1741。

The control of shifting speed

Utilize with other advantages that read gap of reading reagent functionalization be base in gap intrinsic length in conjunction with timeBetween, as discussed in embodiment 13. The subject matter of nano-pore is when apply the bias voltage of enough dominating greatly thermal fluctuation across holeTime DNA displacement high-speed. DNA is with the speed displacement of millions of bases per second, all too fast for the read schemes of any reality. Brantonetal., NatureBiotechnologyvolume28, pp1146-1153,2008 have discussed thisIndividual problem. Use the molecular function in conjunction with DNA base at electrode, a base can keep seizure to reach several seconds.The labor of the AFM data that present in accompanying drawing 59 (Huangetal, NatureNanotechnology,Vol5pp868-873,2010) show, only need to apply little power and improve DNA by receiving by reading reagent functionalizationThe speed in rice hole. For example, the data of accompanying drawing 59 are used to demonstration, and under the 80mV bias voltage across nano-pore, DNA will be with per second 10100 bases that exceed per second are brought up in the speed displacement of individual base under 120mV.

The analysis of polymer

Compound of the present invention, parts, equipment and method can be for analyzing polymers. In some method of operating, partiallyPress, for example, approximately 0.1 to 1V, for example approximately 0.3 to 0.7V, or about 0.5V(V_t) can between electrode, apply by voltage source, attachedVt in Fig. 9. Gap between probe and nano-pore is adjusted by conductor (7 in accompanying drawing 9), until reach establishing of expectationPut an electric current. This can be approximately 1 to 10pA, for example, and approximately 3 to 6pA. For example, can apply displacement bias voltage (V_e) produce DNABy the displacement of CNT. The preferred value of Ve is between 0.1 to 1V.

In some embodiments, one of electrode (for example, probe) can utilize the transversal scanning action of conductor 7Mobile nano-pore of locating the DNA that is successfully shifted on surface, the maximum that gap is conditioned to realize in tunnel signal is distinguished rate.Gap will be set to preferred initial value (for example, the 6pA electric current under 0.5V), carry out little in background tunnel currentAdjust the separation of optimizing from the signal of four kinds of nucleosides.

In analyzing polymers, preferred miscellaneous part has: (a) eliminating at rapid data peak (lower than 40 μ s duration);(b) use the automation peak detection being arranged on higher than the threshold value of 1 to 2 standard deviation of noise level in 0.3s data block; And(c) maintain the adjustment of the servo control mechanism gain of background current signal, thereby frequency response is no faster than 35Hz; (d) obtaining in dataDuring getting, close the device of servo control mechanism. In the time that the servo control mechanism of control mean gap size is retained during data acquisition,Along with servo control mechanism is in response to the order-checking signal adjusting play of expecting, data are distortions. For example,, in accompanying drawing 68,73 and 74Track obtains (the low-response time of utilizing as just having described) in the time that servo control mechanism is opened. Rail in accompanying drawing 70,71 and 72Mark is (there is no SERVO CONTROL) obtaining with fixing gap. Signal is easier to explain. In the instrument with variable gap,Good arrangement is not have the situation down-sampling background signal of DNA, stablizing gap with servo control mechanism, then closing servo control mechanismObtain data, reset gap, then DNA signal stops.

In some embodiments, according to selecting current signal their perdurabgility, in the time of 0.5s or shorter intervalBetween on matching background current digitally, to set up the baseline of identification higher than the peak of this background.

Be appreciated that, further advantage of the present invention is to present and be in little spacing distance (apart from reaching 10Carbon-carbon bond) on hydrogen bond donor and/or any target of acceptor (and/or can the stacking aromatic rings of pi-) can be thisIn scheme, read, if necessary, the signal from destination subset is optimized in adjusting play.

Form the CNT of two electrodes

In other embodiments, CNT can be used to form two electrodes, as attached illustrated in fig. 12. AsFor the CNT of electrode of the polymer that checks order at 61/083,993(" for the establishing based on CNT of the polymer that checks orderStandby ") in described, be incorporated in by reference this. As at Liuetal., Translocationofsingle-strandedDNAthroughsingle-walledcarbonnanotubes.Science,2010,327,pIn 64-67 describe and at 61/083,993(" for the equipment based on CNT of the polymer that checks order ") further describe, utilizing the equipment of setting up on silicon wafer surface, the little gap (referring to accompanying drawing 12) in CNT 30 is crossed in DNA displacement.Open portion by opposing obstacle 35 is to the etched of short duration exposure of oxygen plasma, use conductor 36 bending apparatus, institute subsequentlyState conductor 36 and press film 34, film 34 is bending and be indexed to its side with respect to the fixing point 37 of gap top, canTo make otch very little in CNT. The bending CNT that cuts will fracture it, along with the base quilt that pipe is located is furtherBending, the degree in gap improves. The size in open gap can be passed through from an electrode (31a) to another electrode (31b)The tunnel current passing is measured. In the time having obtained the gap length of expecting (2 to 2.5nm), the end of CNT is with described aboveUrea groups 32 functionalization.

In some method, DNA displacement is by gap 30, and conductor 36 is conditioned to optimize the tunnel current from baseThe separation of signal, described base is passed through in conjunction with urea groups on electrode 31a and 31b across gap.

Embodiment

Synthetic and the sign of embodiment 1. materials

1.1 materials and methods

Proton N MR(¹H) spectrum record on Varian500MHz spectrometer. In chloroform¹H chemical shift is with reference to solventPeak (δ_H=7.26ppm). High resolution mass spec (HRMS) uses atmospheric pressure chemi-ionization (APCI) technology to carry out record. UV lightBe absorbed in record on VarianCary300UV spectrophotometer. Flash chromatography uses the flash chromatography of automation(TeledyneIsco, Inc.CombiFlashRf) carries out. All chemical reagent are purchased from the supplier of business, unless anotherThere is explanation by the use of receiving. 2'-deoxyadenosine and 2 '-deoxyguanosine is purchased from TCIAmerica; Thymidine purchased fromAlfaAesar; 2 '-deoxycytidine is purchased from Sigma-Aldrich. Sure/Seal^TMAnhydrous N in bottle, N-dimethyl formylAmine (DMF) is purchased from Sigma-Aldrich. 1,2,4-trichloro-benzenes (TCB, 99%, Aldrich) in nitrogen on molecular sieve (4)Dry, then decompression distillation after filtration. Every other solvent is by the use of receiving.

1.2 prepare two (tert-butyl group dimethylsilane) (TBDMS) general process of derivative (referring to accompanying drawing 14) of nucleosides

Referring to (D.A.Barawkar, R.K.Kumar, K.N.Ganesh, TetrahedronLetters48,8505（1992）；W.Zhang,R.Rieger,C.Iden,F.Johnson,Chem.Res.Toxicol.8,148（1996）；P.Potier,A.Abdennaji,J.P.Behr,Chem.Eur.J.6,4188（2000）。

Tert-butyl group dimethylsilane chloride (TBDMSCl, 2.5mmol) adds anhydrous nucleosides (1.0mmol), two toIn methylamino pyridine (DMAP, 0.15mmol) and imidazoles (6mmol) solution in dry DMF (10mL). At room temperatureIn nitrogen, stir after reactant mixture spends the night, with saturated water-based NaHCO₃Cancellation, extracts with carrene. Concentrated combinationOrganic layer, residue by silica gel flash chromatography, with 100:0 to the CH of 100:5₂Cl₂–CH₃OH gradient elution carrys out purifying.

3 ', 5 '-bis--O-(tert-butyl dimethylsilane)-desoxyadenossine (1): productive rate 80%.¹HNMR（500MHz,CDCl₃）:δ8.29（s,1H,2-H）,8.09（s,1H,8-H）,6.65（brs,2H,NH₂）,6.41（t,1H,1’-H）,4.56（dd,1H,3’-H）,3.96（d,1H,4’-H）,3.82（dd,1H,5’-H）,3.72（dd,1H,5’’-H）,2.59（m,1H,2’-H）,2.39（m,1H,2’’-H）,0.86（s,18H,（CH₃）₃CSi）,0.05（s,6H,CH₃SiO）,0.03（s,6H,CH₃SiO). HRMS(APCI): to C₂₂H₄₁N₅O₃Si₂+HCalculated value, 480.2826; Measured value, 480.2818.

3 ', 5 '-bis--O-(tert-butyl dimethylsilane)-deoxycytidine (2): productive rate 17%.¹HNMR（500MHz,CDCl₃）δ8.07（d,1H,6-H）,7.14（brs,2H,NH₂）,6.24（t,1H,1’-H）,5.84（d,1H,5-H）,4.38（m,1H,3’-H）,3.92（m,2H,5’-H）,3.77（m,1H,4’-H）,2.42（m,1H,2’-H）,2.08（m,1H,2’’-H）,0.92（s,9H,（CH₃）₃CSi）,0.88（s,9H,（CH₃）₃CSi））,0.11（s,3H,CH₃SiO）0.10（s,3H,CH₃SiO）,0.07（s,3H,CH₃SiO）0.06（s,3H,CH₃SiO）。HRMS（APCI）:C₂₁H₄₁N₃O₄Si₂The calculated value of+H, 456.2714; Measured value, 456.2722.

3 ', 5 '-bis--O-(tert-butyl dimethylsilane)-deoxyguanosine (3): the crude product from chromatography is further logicalCross recrystallization in ethanol (95%) and carry out purifying. Productive rate 21%.¹HNMR（500MHz,CDCl₃）δ13.10（brs,1H,NH）,7.89（s,1H,8-H）,7.11（brs,2H,NH₂）,6.26（t,1H,1’-H）,4.57（t,1H,3’-H）,3.97（t,1H,4’-H）,3.81（m,1H,5’-H）,3.77（m,1H,5’’-H）,2.51（m,1H,2’-H）,2.37（m,1H,2’’-H）,0.91（s,9H,（CH₃）₃CSi）,0.90（s,9H,（CH₃）₃CSi））,0.10（s,6H,CH₃SiO）0.07（s,6H,CH₃SiO）.HRMS:（APCI）C₂₂H₄₁N₅O₄Si₂+HCalculated value, 496.2775; Measured value, 496.2767.

3 ', 5 '-bis--O-(tert-butyl dimethylsilane)-thymidine (4): productive rate 83%.¹HNMR（500MHz,CDCl₃）δ9.78（brs,1H,NH）,7.40（s,1H,6-H）,6.27（t,1H,1’-H）,4.33（t,1H,3’-H）,3.85（t,1H,4’-H）,3.80（dd,1H,5’-H）,3.69（dd,1H,5’’-H）,2.18（m,1H,2’-H）,1.93（m,1H,2’’-H）,1.84（s,3H,5-CH₃）,0.85（s,9H,（CH₃）₃CSi）,0.82（s,9H,（CH₃）₃CSi））,0.04（s,6H,CH₃SiO）0.00（s,6H,CH₃SiO）.HRMS:（APCI）C₂₂H₄₂N₂O₅Si₂The calculated value of+H, 471.2711; Measured value, 471.2712.

The preparation of 1.3 stock solutions

There is the saturated solution of the nucleosides (dA, dG, dT, dC) of the oh group of tert-butyl dimethylsilane group protection(1.0mg) add 1,2 of new distillation to, in 4-trichloro-benzenes (20ml), in ultra sonic bath ultrasonic 10 minutes. Solution filter paper(1#, Whatman) filters, and is kept in glove box (moisture is lower than 0.5ppm, and oxygen is lower than 0.5ppm). Working solution is logicalCross and prepare with TCB dilution stock solution.

The concentration of 1.4 stock solutions

Overlapping due to the UV light absorption of TCB and nucleosides, the concentration of stock solution is measured by exchange of solvent. In vacuumUnder 80 DEG C of aliquot from stock solution (1ml), remove TCB, residue is dissolved in the chloroform of same volume again, measureConcentration is determined in its UV light absorption.

Utilize respectively the series of dA, dG, dT and dC dilution, under their maximum absorption wavelength, measure institute in chloroformThere is the UV extinction coefficient of nucleoside derivates. Dilution factor from 3.5 to 200. Curve carries out in Origin8. Stock solutionUltimate density in following table, list.

The saturated concentration of nucleosides in table 1.TCB stock solution. Bottom line has been listed the final concentration using in tunnel survey(each nucleosides being produced to the concentration of approximately equalised read rate).

Embodiment 2: probe and surperficial Preparation and characterization

Etch gold (S.Changetal., Nanotechnology20,075102(2009)) (AlfaAesar,0.25mmdiameter, 99.999%pure) and Pt(20%Ir) (L.A.Nagahara, T.Thundat, S.M.Lindsay, Rev.Sci.Instrum.60,3128(1989)) probe, and preparation surface (J.A.DeRose, T.Thundat, L.A.Nagahara, S.M.Lindsay, Surf.Sci.256102(1991)) in hydrogen flame, move backFire.

0.3mg benzoic acid is dissolved in the 2mLN by argon-degassed, dinethylformamide ((Sigma-Aldrich, >99.99% purity) in. After hydrogen flame annealing, substrate is directly immersed this solution two hours, then uses N, N-dimethyl formylAmine, acetone and 1,2,4-trichloro-benzenes clean, and use front at mobile N₂In dry.

Before modifying, probe is at piranha(H₂SO₄/30%H₂O₂, 3:1: produce heat and oxygen, handled) in clearClean. Then they immerse in 1mM benzoic acid solution and spend the night, and with DMF, acetone, 1,2,4-trichloro-benzenes is clearClean, before using, dry up. All measurements are carried out in the neat solvent of freshly prepd, nucleosides or solution.

Surface ellipsometry, STM(A.H.Sch fer, C.Seidel, L.Chi, H.Fuchs, Adv.Mat.10,839(1998)) and FTIR(S.E.Creager, C.M.Steiger, Langmuir11,1852(1995)) characterize. FTIR spectrum has clearly illustrated that benzoic acid part is exposed, and neutral form in it. Measure the back of the bodyScape tunnel signal shows in accompanying drawing 2.

2.1 polarization current and electrochemistry seepage

The absolute value of peak point current is subject to the impact of electrochemistry seepage, as follows: use away from surperficial probe measurement at rollbackAfter background leakage current, tunnel current is set. If this is substantial (tens of to hundreds of pA),, even if probe is not(thereby seepage produces on their whole surface) of insulation, in the time that probe is taken to surface, around changes as tips of probesThe result of the diffusion rate becoming, seepage still can change (with pA level). Thereby, the seepage application to the probe away from surperficialThe seepage near surperficial probe can be excessively corrected in correction. As a result, apparent tunnel current is by overestimate, if seepage is in oneDifferent between nucleosides solution and another kind of nucleosides solution, change the true set-point of nucleosides and nucleosides. This effect is enough largeTo changing the apparent order of magnitude (table 1) that is in dC and dG peak under saturated concentration. Be diluted to the working concentration that table 1 shows, 0.5V is inclined to one sideThe leakage current of depressing is 1.0 to 2pA(dA), 0.0 to 1.0pA(dT) and 0.3 to 1pA(dG). The in the situation that of dC(0.8 μ M), originally observes the electric current of 15pA, but this is reduced to several pA after one of solution hour is exposed. TheseBackground from deducting the baseline tunnel current of this report. They seem not cause appreciable error, by single nucleosidesSimilitude between data and the data of mixture proves. The example of the initial data of dT, dG and dC can be in accompanying drawing 15Find.

The gain of 2.2STM servo control mechanism

The frequency response of servo control mechanism is not by relatively there is no (accompanying drawing 16A) and having (accompanying drawing 16B) to apply the 1/f of servo control mechanismNoise curve is determined.

Current track is the Fourier that transforms and show as spectral density, according to:

(formula I)

Wherein n is channel quantity (Δ t=20 μ s, N=50000). Solid line in accompanying drawing 16A is the matching to 1/f. WatchingWhen taking loop and closing (accompanying drawing 16B), noise data is suppressed to lower than 35Hz, corresponding to the 28ms response time. This is enoughLong and can distortion all pulses except long pulse (long pulse in the illustration of accompanying drawing 15 has shown peak electricityThe little reduction of flow horizontal, consistent with the servo control mechanism response of measuring).

The peak of 2.3 automations detects

For speed and elimination operator deviation, data analysis is automation. If run into extremely noisy background (dirtThe feature of dying), be the tranquiler region that probe is moved to substrate to process operator's input.

Principle challenge is the low-frequency instability in background current, and it can not fully be proofreaied and correct by servo control mechanism. IfFor the acceptance at peak is used little fixed threshold, even also produce a large amount of falsenesses higher than the very little baseline variation of this threshold valueCounting. Overcome as follows this difficult problem.

Electric current-time data (obtaining under 50kHz) is broken into the piece of 0.3s. Amplitude in piece is by binned, dataLower Half carry out matching by Gaussian, it is to check that the mean value of Gaussian equals the program of the base current of expecting.The HWHH of Gaussian is used to determine SD, the σ of baseline noise. It is higher than 0.3 by threshold value setting that the data that herein show are passed throughNoise 2 σ in s data run. Because noise level changes during the perdurabgility of operation, this variable threshold value has caused changeThe sieve of changing cuts, in the time that data are aggregated, this may change the distribution shape that minimum current reads (, for dT and with one nakedReveal the data that electrode obtains). At data (dT, 4.3 μ M, G_bl=12pSV=0.5V) (100 0.3 of 30s operationsS fragment) top sieve the acting in accompanying drawing 17 of three kinds of selections of cutting shown.

Very short pulse (in the limit of instrumental resolution) can occupy the main of data, seems the not body to nucleosidesPart is responsive. Thereby only one ((40 μ whole spikes s) are eliminated 20 s) or two of μ data points perdurabgility. Spike lifeBeing distributed in accompanying drawing 27-29 of time provides.

The data that 2.4 use bare electrodes obtain

With bare electrode obtain data in accompanying drawing 18 and 19, show. Accompanying drawing 18 and 19 has shown that it is asymmetric distributing.Our putative molecule configuration is random distribution, and tunnel current is responsive exponentially to the change of position. Thereby, weIn the logarithm of electric current, use Gaussian distribution:

(formula II).

As shown in the curve in accompanying drawing 18 and 19, the equation and the data that in formula II, show are good fit. Use from accompanying drawing 19AThe double-log plot of data represented the quality of matching, in accompanying drawing 20, show. Curve is parabola.

Table 2: process is arranged on the peak current of four kinds of nucleosides of the exposed gold electrode under 20pS and 40pS electrical conductivity（I_p), distribute high electric current side on width (I_0.5 ⁺) and read rate (RR) (bias voltage=0.5V).

For the narrower distribution recording with the probe of functionalization, poor between Gaussian and the matching of Gaussian logarithmDifferent is more inapparent, although be still better than significantly and the matching of Gaussians with the matching of Gaussian logarithmic function. GreatlyMost data and two be Gaussian's and matching, and second concentrates on the twice place of first current peak:

(formula III)

For narrower distribution, HWHH obtains by following being similar to:

(formula IV).

The data of the mixed solution of 2.5 nucleosides

The data of the mixed solution of nucleosides provide in accompanying drawing 21. The reading result of the diaphragm mixing is slightly different,Indicate lip-deep being separated. The distribution showing in accompanying drawing 3F and accompanying drawing 3H, accompanying drawing 5C and accompanying drawing 21 is by six notWith some effects on surface sampling, and add that data obtain. For 0.24 main body dA:dG concentration ratio (table 1), the peak of measurementThe ratio of area is 0.6, shows that dA is with the compatibility mating surface larger than dG. As main body concentration ratio (bulkConcentrationraitio), while changing into dA/dG=0.12, the ratio of peak area is only reduced to 0.4, has indicated mixtureComplicated adsorption isotherm.

For 0.19 main body dT:dC concentration ratio (table 1), the ratio of the area at corresponding peak has indicated the surface of 1:1 denseDegree ratio, shows that dC has much higher compatibility to this surface. In the time that concentration ratio is changed into 0.09:1 (accompanying drawing 21), integrationPeak area shows, surface concentration ratio is changed into dT/dC=0.2.

Thereby for dC/dT mixed layer, compared with in the situation of dA/dG mixture, larger in apparent surface's concentrationMany changes are caused by the change producing in main body concentration, supposition is different solvent compatibilities, different surfacesCompatibility, the result of the competition between the interaction between nucleosides from the teeth outwards. However, relevant to the composition loweringUniformity ground, peak has reduced, and has verified that we distribute at the peak based on pure nucleosides solution current measurement value.

The calculating of the electrical conductivity of 2.6 hydrogen-bonded compounds

Because the electric current assessment of applied bias voltage utilizes, ballistic transport is theoretical to be determined. The electronic state of gold is crossed over molecule and is let outLeak, produce tunnel current. Between the transmission period of electronics, the elasticity inscattering (In-elasticscattering) of electronics is not examinedConsider. Determine electronic current by electronics under the fermi level of metal through the transfer function of molecule. Only consider very littleBias voltage (+/-0.1V). In this region, I-V characteristic is all linear, thereby, characterize simply knot by its electrical conductivityReally. Electrical conductivity equals the product of the transfer function under amount and the fermi level of electrical conductivity.

The calculating of transfer function provides (J.K.Tomfohr, O.F. by the standard results from scattering theorySankey,J.Chem.Phys.120,1542（2004））,, itsMiddle E is energy, (fermi level of golden contact), and Γ is the spectral density of the state of left side and right side metal contact, and G_MFor molecule Green ' s function propagator. Euler integral of the second kind contain metallic state and they how with the full detail of molecular tie, G_MThe full detail that contains electronic state in molecule. Green ' s function propagator will arrive metal contact along metal contactPath is with seemingly decay exponentially of near distance.

For spectral density and Green ' the s function of computing mode, people need model and the modeling half of electronic stateThe method that unlimited metal leads. We be most advanced and sophisticated and substrate and semi-infinite flat gold (111) surface construction model.

Connect sulphur atom at the end of molecule and be better than the hollow site of Au. Use superlattices slab geometry. This meaningThe system of wearing is the periodic array of the golden slab (being originally thin) with sandwich molecule in ad hoc structure. That repeats is superLattice structure is like this, thereby Bloch ' s theorem can be for measuring the electronic state of whole system. The electronics of whole superlatticesStructure is as one man measured by oneself in Density Functional Theory. In order to proofread and correct the fact that slab is 5-7 layer Au, by the middle of selectingLayer represents main body gold, and them are extended to infinity by a kind of method of recurrence.

The local Atomic Orbits of use fireball (O.F.Sankey, D.J.Niklewski, Phys.Rev.B40,3979(1989)) type is determined electronic structure. Partial orbit has limited radius, thereby is excited from ground state very slightly.SIESTA code (P.Ordejon, E.Artacho, J.M.Soler, Phys. in Density Functional Theory, are usedRev.B53,10441(1996)). All atoms are described with pseudo potential, and it has eliminated all kernel states. For instituteThe baseset that has atom to use is that double zeta adds polarization (DZP), but Au uses single zeta to add polarization (SZP).

The multiple different geometries of combination between two readers and target base are explored. In all cases, makeWith the lax DFT geometry of reader and base. The limited set whole molecular system that relaxed calculating. In table 3The result of report has been used the geometry of lax individual molecule, and then individual molecule is converted in accompanying drawing 1A-D rigidlyThe structure of combination. Important variable is that (it is for example set to this hydrogen-bonded according to experiment and DFT to hydrogen key lengthThe desired value (M.H.Lee, O.F.Sankey, Phys.Rev.E79,0519111(2009) of molecule)), andDistance between metal leads. Lead distance of the metal of estimating is set to according to tunnel decay coefficient and by solventThe value that background tunnel current is estimated.

Between theoretical and experiment quantitative inconsistent (table 3) show as very large, particularly like this for the situation of dT. SoAnd, ignore the tunnel-effect of solvent mediation and may ignore important extra current, be equivalent to only use the electrode of a functionalizationDetect, this wherein top contact be solvent mediation. This is a kind of significant electric current, should add to as a setting at thisThat calculates passes in the value of key. The second source of error may come from our estimation to tunnel gap. Little estimationHigh (0.1nm in 2.5) are by causing the remarkable reduction of tunnel current of calculating, due to the very large electronics of the hydrogen bond extendingDecay coefficient. M.H.Lee, O.F.Sankey, Phys.Rev.E79,0519111(2009).

Embodiment 3: tunnel survey

We carry out on the PicoSPM scanning probe microscopy (Agilent, Chandler) that is connected in digital oscilloscopeTunnel measurement. In the time that 4-mercaptobenzoic acid functionalization is all used in probe and gold (111) substrate, the tunnel background signal pair in TCBRelatively muting in set-point electric current, I under 0.5V bias voltage_bl10pA at most, electrical conductivity 20pS(accompanying drawing 2). By nucleosidesSolution is placed in liquid lattice, after polarization, electric current is down to a little value, and we are providing low noise background signal previouslyAgain access probe under tunnel current level. In tunnel signal, current spike is obvious (accompanying drawing 15) immediately. Due to nucleosidesSurface concentration and gap in the efficiency of molecule trapping be all previously known, the concentration that we adjust nucleosides solution obtainsApproximately equalised in tunnel gap " spike rate " (table 3).

	dT	dG	dC	dA
					The G(pS measuring)	13.6±0.3	18.6±0.9	25.3±2.5	33±1.9
The G(pS calculating)	0.04	0.12	0.51	1.05
					Read rate (s-1）	7.1±1.4	5.5±1.1	5.5±1.1	6.6±1.3

Table 3: in I_blThe electricity with calculating of measuring during the tunnel of the functionalization under=6pA, V=0.5V connects is ledRate. The value of measuring is the mean value (error is ± 1sd) of three independent operatings. The electrical conductivity of calculating is for as in accompanying drawing 1A-DThe structure showing. For the nucleosides concentration between 0.8 to 4.3 μ M, read rate is based on the counting obtaining in the 180s cycle.Inconsistent the ignoring of may reflecting background contribution of number range between theoretical and experiment, it is via entering an electrodeThe tunnel of the solvent mediation of the molecule of upper combination. Absolute value will be subject to the impact of the inaccuracy in gap length estimation.

Many " spikes " have shown the combination of individual molecule and " thump telegraph repeater " feature of unconjugated dual intensity level in gap(S.Changetal., Nanotechnology20,075102(2009)) (illustration, accompanying drawing 15). STM is set servoGain makes the spike of maximum length in time only be subject to the effect (accompanying drawing 16) of Current Control servo control mechanism.

The distribution that we utilize the program of customization to produce peak current, analyzes the height of spike. Program catches at baselineThe upper signal higher than two standard deviations of noise, also eliminates on the time only one or two point (, reaching for 40 μ s duration)Data. Measure distribution on filtration parameter selection act on accompanying drawing 17 and accompanying drawing 27-28 in show. Accompanying drawing 3 showsThe distribution of these measurements how to be subject to the impact of the functionalization of electrode. Be distributed in accompanying drawing 3A and accompanying drawing 3C with bare electrode recordMiddle demonstration. In order to use bare electrode tracer signal, we must be by operating and reduce slightly between tunnel under the electrical conductivity of 20pSGap. Even in this less gap, to pyrimidine nucleoside with bare electrode read compare reading of purine nucleosides be frequency moreMuch lower (accompanying drawing 18, table 2). As shown in accompanying drawing 18-20, the Gaussian of the logarithm by electric current distribute (solid line),The CURRENT DISTRIBUTION matching well of measuring. (dA is 15.9 ± 0.4pA to the peak current difference of the matching of these two kinds of nucleosides, and dG is18.7 ± 0.2pA), but difference (2.8pA) is less than the width (~ 15pA) distributing in high electric current side. When the gap of improvingWhen the substrate of place (corresponding to 12pS) use functionalization and exposed Au probe duplicate measurements, the distribution of the electric current of measuring is by quantityLevel ground (accompanying drawing 3B-dA) (the accompanying drawing 3D-dG) that narrowed, but peak current is significantly not different. For two be all bare electrode andThe electrode of a functionalization, the distribution in spike life-span is quite similarly (accompanying drawing 29). Thereby, seem likely, with nakedThe spike that electrode is observed is also corresponding to the instantaneous bonding state of nucleosides. If this is actual conditions, use the electrode of functionalizationNarrowing of observing must be the result of the minimizing of bonding state number of types in tunnel gap. When two probes and substrate are all by meritEnergyization time (accompanying drawing 3E – dA, accompanying drawing 3G – dG), the peak current of dA is apparently higher than the peak current of dG. Thereby, when two electrodes allCan while being functionalized, produce distinctiveness signal, but they come from single nucleosides? " thump telegraph repeater " signal is singleThe feature of individual molecule reading result, distributes to small size (accompanying drawing 3B, accompanying drawing 3D, the accompanying drawing 3E, attached at the peak of bimolecular reading result" 2 " in Fig. 3 G) to show once to exceed reading of a molecule be rare. But electrochemical leakage current can be introduced and getCertainly in the current error of nucleosides, thereby the electric current of measuring may be separately from individual molecule electric current. The accuracy that tunnel readsBetter test can carry out with the mixture of two kinds of nucleosides, thereby because any error of electrochemistry background all existsIn two kinds of set of signals. Accompanying drawing 3F has shown the CURRENT DISTRIBUTION obtaining with the mixture of dA and dG. Higher current peak is located substantiallyAt the same current place to independent dA record, thereby should count the dA molecule in this mixture. By dividing dA in solution equallyConcentration confirmed this distribution (accompanying drawing 3H). Surface concentration is unknown in advance, depends between nucleosides that effects on surface is in conjunction with positionThe competition of point, and get back to the different dissociation rate in solution, thus absolute signal rate can not be separated quantitatively with regard to concentrationRelease. Most of data in this picture well matching suppose single molecule reading result, only 5% reading result withIn gap, there is dA consistent with dG (" dA+dG ", accompanying drawing 3F) simultaneously.

DC and dT are observed to the feature (accompanying drawing 5 and accompanying drawing 29) of same type, but exposed gap and exposed baseThe data at the end must gather (20pA, corresponding to 40pS) under larger tunnel current, phonetic to obtain (size is less)The reading result (accompanying drawing 19) of the remarkable quantity of pyridine nucleosides. In the time reading with the probe of functionalization, dC and dT in the sample of mixingAlso be (accompanying drawing 5C and the accompanying drawing 21) obviously separating.

Under given bias voltage, the absolute value of peak current is directly proportional (accompanying drawing 7A) to the baseline electrical conductivity in gap, that is, it withThe reduction of gap improves exponentially, is similar in larger tunnel gap, other hydrogen-bonded systems to be reported(S.Changetal., Nanotechnology20,075102(2009)). We have found at fixing gap lengthThe interested dependent evidence (, gap electrical conductivity) (accompanying drawing 22) of lower peak current to bias voltage, show with two electrodes allIn conjunction with molecule non-linear current-voltage-dependent may. Reading frequency also improves (accompanying drawing 24) along with gap turn narrow.On the other hand, the part of polymolecular reading result improves rapidly (accompanying drawing 7A and accompanying drawing 25) in less gap, thereby seesThe 12pS that gets up is the optimum value of baseline electrical conductivity under 0.5V bias voltage.

At I_blThe value of the peak current of measuring under=6pA, V=0.5V is represented by cross-hatched post in accompanying drawing 7B. ThisThree the different operations (being undertaken by different team from sample preparation to data analysis) to each of four kinds of nucleosides a bitResult. The peak of every kind of nucleosides is by separating with the comparable amount of width distributing, allowing as two " well " and contactThe fragment of unimolecule reading result identify base (accompanying drawing 26) with p >=0.6.

We also use the substrate of functionalization and the post of the dark-coloured shade of exposed Au() or the post of exposed Pt(light color shade)Probe records data. It is very little that peak current changes between nucleosides and nucleosides, is the resistance R relevant to exposed contact_cExpected result (X.D.Cuietal., Science294,571(2001)), but optionally lack can not be onlyIllustrated by independent contact resistance. If we suppose that the reading result of two functionalize tips determined the electricity of individual moleculeResistance, R_m, the resistance of the connection of a bare electrode should be by R_j=R_c+R_mDraw. Accompanying drawing 7B has shown, an exposed goldThe signal of electrode is insensitive for the resistance of molecule, and the sensitiveness of exposed Pt electrode is simple " series resistance " mouldIt is only about half of that type predicts the outcome, and may reflect the mode (V. that affects the position of molecular state with the combination of electrodeMeunier,P.S.Krsti?,J.Chem.Phys.128,041103（2008）。

Under 12pS electrical conductivity, we estimate that gap is about 2.5nm, utilize, whereinG₀The quantum of electrical conductivity (77 μ S), β=6.4nm^-1（J.He,L.Lin,P.Zhang,S.M.Lindsay,NanoLetters7,3854(2007)). What accompanying drawing 1A-D had shown that we think has two electricity that are all functionalizedIn the gap of the utmost point, most probable hydrogen bonding (energy minimization) structure of four kinds of nucleosides. We have carried out this four kinds of moleculesThe Density functional calculations of the electrical conductivity connecting, the electrical conductivity of prediction is listed hereinafter, and the value of measurement is listed in table 3. Electricity is ledThe order of magnitude of the prediction of rate conforms to experiment, but absolute value is significantly lower, may be the mistake due to tunnel gap sizeHigh estimation. The lifetime data (accompanying drawing 29) of dT has shown the minor alteration in the time that two electrodes are all functionalized, thus dT spikeCan represent the tunnel of an electrode place solvent mediation. Because solvent molecule is not included in simulation, this extra tunnelIn roadization contribution prediction, do not have. Thereby constant background should be added in the electric current of each prediction, this will reduce predictionAnd measure current range between deviation.

Synthesizing of embodiment 4:4-sulfydryl benzamide

The synthetic of 4-sulfydryl benzamide carries out according to following scheme:

。

4.1Materials and methods

Proton N MR(1H) spectrum on Varian400MHz mass spectrograph at 400MHz record, or at Varian500MHzOn mass spectrograph at 500MHz record, the NMR(13C of carbon) spectrum on Varian400MHz mass spectrograph at 100MHz, orOn Varian500MHz mass spectrograph at 125MHz record. Use atmospheric pressure chemi-ionization (APCI) technology to obtain HRMS spectrum.Flash chromatography is at CombiFlashRf(TeledyneIsco, Inc.) in carry out. Except as otherwise noted, all reagent purchased fromAldrich。

4.2 step 1:4-trityl mercaptobenzoic acids

4 ?mercaptobenzoic acids (1.54g, 10mmol) and trityl chloride (2.79g, 10mmol) are dissolved in DMF(25mL), in, stir at ambient temperature 36h. Decompression is lower to desolventizing. Residue is dissolved in chloroform (50mL), washes with waterWash (3 × 25mL). Organic layer is dry on MgSO4, filters and concentrates. Obtain compound 1(3.20g as white solid,81%）。1HNMR（500MHz,CDCl3）:7.67（d,2H）,7.21‐7.39（m,15H）,6.99（d,2H）。

4.3 step 2:4-trityl sulfydryl benzamides

Ammonia (0.5M, 1mmol in dioxane) is added drop-wise to compound 1(198mg, 0.5mmol at 0 DEG C), 1-hydroxyl-BTA (HOBt) (68mg, 0.5mmol) and 1,3-dicyclohexyl carbodiimide (DCC) (103mg, 0.5Mmol) at THF(5mL) in solution in. The mixture of allowing generation is warming to room temperature, stirs 24h. After filtering, filtrateWith saturated NaHCO3 solution washing. Organic layer is dry on MgSO4, filters and concentrates. Residue is by flash chromatographic purifying(silica gel, carrene: methyl alcohol gradient is from 100:0 to 100:3) produces the compound 2(154mg of white solid, 78%). 1HNMR（400MHz,CDCl3）:6.98‐7.43（m,19H）,5.95（brs,1H）,5.75（brs,1H）；HRMS(APCI+): measured value, 396.1442; C26H22NOS+H calculated value, 396.1422.

4.4 step 3:4-sulfydryl benzamide

Compound 2(60mg, 0.15mmol) be dissolved in trifluoroacetic acid (TFA) (2mL) and triethyl silicane (TES) (2mL)Mixture in, at room temperature stir 2 hours. Solution under reduced pressure rotary evaporation is dried. Residue is from hexane and dichloroIn the mixture of methane (V:V=1:1), crystallization produces the compound 3(12mg of white solid, 52%). 1HNMR(500MHz,CDCl3）:7.68（d,2H）,7.31（d,2H）,6.50（brs,1H）,6.29（brs,1H）,3.61（s,1H）；13CNMR（125MHz,CDCl3）:169.9,138.0,129.4,128.5,128.2.4HRMS（APCI+): measured value, 154.0326; C7H7NOS+H calculated value, 154.0326.

Embodiment 5: generation, functionalization and the sign of electrode

5.1 electrodes produce

Gold tip utilizes mixture (volume ratio 1:1) electrification from gold line (Aesar99.999% is pure) of HCl and ethanolLearn etching. Only select sharp-pointed tip (using the light microscope of 300X magnifying power to judge) for insulation process. High-density polyethyleneAlkene (HDPE) is as insulator. Before insulation, the most advanced and sophisticated mixture with piranha(oxygen peroxide and sulfuric acid of gold, volume ratio1 to 3, careful: this material is may explode with the reacting of organic material) the clean organic pollution of removing for 1 minute, and useDistilled water, ethanol clean,, dry up with the nitrogen compressing. During insulating, HDPE is coated with instrument at 250 DEG C at homemade tipOn device, melt. The infiltration of the HDPE of fusing is on-insulated by covering most advanced and sophisticated most of region with insulating materials, only leaving top. The surface area of the most advanced and sophisticated exposure of insulation characterizes by the cyclic voltammetry in the potassium ferricyanide. Survey by cyclic voltammetryExamination insulation and uninsulated tip. The tip of insulation provides more consistent and regular electrode. Accompanying drawing 30 and 31 has illustratedThese results. Accompanying drawing 30 has shown the cyclic voltammetry (electromotive force vs.Ag line) of gold line exposed in the 50mM potassium ferricyanide.Accompanying drawing 31 has shown the cyclic voltammetry at the coated STM tip of HDPE. Suppose pointed shape and use formula that hemispherical exposesImax=2 π RnFCD, the typical exposed surface area of coated scan-probe is on the order of magnitude of 10-2 μ m.

5.2 functionalization

Gold substrate anneals to remove the Au surface of depolluting and forming well-ordering in hydrogen flame. The 4-preparing according to embodiment 4Sulfydryl benzamide dissolves (1mM) in methyl alcohol, avoids the oxidation of mercaptan by argon-degassed. The substrate of dielectric tip processingImmerse in this solution 2 hours. This causes the formation of the upper benzamide individual layer in surface. The functionalization time spy of having degraded extendingInsulator on pin, thus the processing of probe is limited to 2 hours. The functionalization of carrying out gold substrate reaches 20 hours.

After deposition, the thickness of molecule SAM passes through ellipsometry (Gaertner, Skokie, IL) at 632.8nmIncidence angle with 70 degree on wavelength is measured. The optical constant (200nm thickness on mica) of the exposed gold substrate of new hydrogen flame existsMeasure before the deposition of molecule, n=0.2, k=?3.53. SAM optical constant is set to nf=1.45 and kf=0. 4-sulfydryl benzeneThe thickness of formamide individual layer is measured as 0.70 ± 0.17nm.

The infrared absorption spectroscopy of SAM uses ThermoNicolet6700FTIR(ThermoFisherScientific, MA) attached SmartAperturedGrazingAngle carrys out record. Use SmartOrbit(rhombusSingle-hop ATR annex) gather the spectrum of powder sample. Accompanying drawing 32 has shown 4-sulfydryl benzamide individual layer (line of below) and powderThe FTIR spectrum at end (line on top). In individual layer IR, 3487cm ?1,1610cm ?1 and 1404cm ?the absworption peak at 1 placeBe assigned to respectively discontinuous NH2 N ?H extend (there is no hydrogen bond), acid amides band I and acid amides band II.

Elliptical light degrees of data has pointed out almost completely individual layer to cover, however STM image shown (accompanying drawing 33) cover withPatch formula occurs. The read rate recording in paper is to be positioned at the reading result that the probe above the patch of functionalization gathers. ?In accompanying drawing 33, STM image has shown the island of sulfydryl benzamide on gold (111) surface. (, 0.5 volt tip most advanced and sophisticated at goldBias voltage, the image in the 1mMBP buffer solution of 10pA set-point. )

5.3 electrode imagings

Characterize electrode by light microscope and transmission electron microscope (TEM) image. Accompanying drawing 34a-c has shown naked electricityThe utmost point, accompanying drawing 34d-f has shown the tip that polyethylene is coated. Accompanying drawing 34a has shown typical " good " most advanced and sophisticated light microscope figureResemble. This tip further characterizes by transmission electron microscope (TEM), as accompanying drawing 34b-c shows. In this caseTip radius is about 16nm(accompanying drawing 34c). Carbon-coating (covering the most advanced and sophisticated white layer of gold) deposits during TEM imaging. Dash lineArc has the radius of 16nm. The radius of measuring generally arrives in the scope of 20nm 5. Tip end surface is normally level and smooth, stillSometimes observe projection (height 1-2nm). At first sight surprisingly, this " blunt " probe can produce unimolecule and dividesDistinguish, (be even better than using functionalization but molecule absorption has from the teeth outwards produced the local high point of can unimolecule differentiatingThe resolution of interior molecules structure of AFM probe illustrated).

The light microscope image of typical dielectric tip shows in accompanying drawing 34d. This tip is not aobvious in experimentShow leakage current (lower than the measuring limit of ~ 1pA) and about 8pA(peak to peak, that is, higher than baseline 4pA) 120Hz noise. PhaseShow in accompanying drawing 34d-f with most advanced and sophisticated TEM image. Arrow in accompanying drawing 34e-f has shown that the gold exposing is (due to coated electricityLotus, high-resolution imaging is impossible).

Embodiment 6: the sign of tunnel gap

6.1 water and buffer solution

The tunnel gap with electrode of describing in embodiment 5 is used distilled water and 0.1mM phosphate butter (PB – pH=7.4) characterize. From independent buffer solution, observe little signal with bare electrode, but when two electrodes be all functionalized,Tunnel gap electrical conductivity is arranged on 20pS or they are much rarer when lower. Referring to accompanying drawing 55. With the feelings in independent waterCondition (β ~ 6.1 ± 0.7nm^-1-¹¹) compare, use more faster (decay normal of two tunnel electrode decay being all functionalizedNumber, β=14.2 ± 3.2nm^-1). The embodiment 6.2 vide infra. Estimate the tunnel under i=10pA and V=+0.5VGap is just over the length (, being a bit larger tham 2nm) of two benzamide molecules.

6.2 background water signals

Use the gap length of 0.5 volt of lower 20pS, the control experiment of bare electrode in the substrate of functionalization in distilled waterObtain background thump telegraph repeater signal by a small margin (about 6pA under 0.5 volt of bias voltage, accompanying drawing 35). But, use functionalizationThe tip of suprabasil functionalization, such signal be generally do not observe (signal observed result once in a while may be fromThe covering of incomplete 4-sulfydryl benzamide on the surface of tip or substrate). These background signals can be by data analysisIn threshold value get rid of because they are less in amount, and more uncommon than DNA signal.

6.3 tunnel attenuation curves

Attenuation curve is measured with being combined in distilled water of functionalization and non-functional polarizing electrode. Attenuation constant (β) is according to Ln(I) slope of the linear fit of the plot of vs. distance calculates (accompanying drawing 36). Accompanying drawing 36 has shown the tunnel electricity in pure H2OStream attenuation curve (marking and drawing in each case multiple curves). Accompanying drawing 36a has shown exposed gold electrode. Accompanying drawing 36b has shownTwo electrodes that are all functionalized. Accompanying drawing 36c has shown the electrode of an exposed electrode and another functionalization. In large distanceSignificantly improving from the electrode detection of upper functionalization to β, shows that the variation of the gap composition that we carry out is mutual from benzamideThe region of effect is converted to their noninteracting regions. Being distributed in accompanying drawing 37 of the value of the measurement of β shows. Accompanying drawing 37 is aobviousShow the histogram of β in pure water, (a) exposed gold electrode; (b) all use two electrodes of sulfydryl benzamide functionalization; (c)An electrode is functionalization, and another is exposed. Gaussian matching (mean value ± SD) produces: (a) 6.11 ± 0.68nm^-1（b）14.16±3.20nm^-1（c）6.84±0.92nm^-1。

Embodiment 7: analyzing DNA nucleotides in tunnel gap

DNA nucleotides (10 μ M in PB) imports to and uses the electrode of describing in embodiment 5 to produce in aqueous electrolyte solutionIn raw tunnel gap. These nucleotides have produced the characteristic noise spike shown in accompanying drawing 56c-f. Signal-count rate is (attachedIn Figure 57, limit) from 25 counting/s(5-methyl-deoxycytidine 5'-monophosphates, d^mCMP) change to significantly and be less than 1c/S(deoxycytidine 5 '-monophosphate, dCMP). Use thymidine 5' monophosphate (dTMP) not to be recorded to signal completely, signal has been seenResemble contrast (accompanying drawing 55) completely. STM image shows, this nucleotides is tied very consumingly with surface (and being speculated as probe)Close the interaction that blocking-up individual molecule is crossed over contact.

There is (having shown below longer signal operation) in electric current, the distribution of spike height is very good in the outburst of spikeTwo Gaussians of ground matching logarithm distribute, as accompanying drawing 56g-j shows (fitting parameter is described in the following Example 8).To exceeding the step-by-step counting of SD1.5x of local noise background, generally produce that these are straight by only higher than the pulse of 6pASide figure (the complete description of analytic process is provided by Changetel.).

DCMP has produced the highest signal and minimum counting rate, and desoxyadenossine 5 '-monophosphate (dAMP) and d^mCMP producesMinimum signal and the highest counting rate are given birth to. As discussed in following examples 9, cytidine and 5-first in organic solventBetween base cytidine, find Light Difference. There are three kinds of bases (dAMP, d that narrower pulse height distributes^mCMP and GMP) normalThe normal outburst that shows " thump telegraph repeater ", this is the feature (remarkable especially for dAMP) in the source that rises and falls between two energy levels.It is the strong instruction that the individual molecule held back during tunnel signal is connect by tunnel produces that such dual intensity level distributes. From nucleotidesThe feature of Tunnel Noise in table 4, summarize.

Table 4: nucleotides Tunnel Noise characteristic. Parameter defines in accompanying drawing 57

* the error in the matching of exponential distribution.^§Standard error.

Aspect spike amplitude and aspect their the time distribution of signal, dAMP signal and dCMP signal are wellSeparate d^mCMP signal separates (table 4 and below discussion) well with dCMP signal. For this reason, further in embodiment 11Research by A, C and^mThe DNA oligomer of C base composition.

Embodiment 8:Gaussian and CURRENT DISTRIBUTION matching

The peak producing from the data of embodiment 7 the logarithm of tunnel current with Gaussian fitting of distribution, be a kind of falseDetermine the model that the random distribution of tunnel geometry is sampled exponentially. For the current data in water, need two peaks,Mean that two kinds in conjunction with geometry:

Equation S1.

Fitting parameter is listed in table S1.

Table S1: intensity distribution fitting parameter.

Embodiment 9: the CURRENT DISTRIBUTION of cytidine and 5-methylcytidine in organic solvent.

In organic solvent, measure with electrode and 4-sulfydryl benzamide reader according to embodiment 4-5^mThe data of CBe included in this, overlap in organic solvent more much biggerly to show between the signal of these two kinds of bases, this has represented is working asIn front work, hydrone is playing a role aspect the unlike signal of generation and C and meC. Accompanying drawing 40 has shown cytidine (solid line)With^5meThe CURRENT DISTRIBUTION that cytidine (short-term) uses benzoic acid reader to measure in trichloro-benzenes solvent.

Embodiment 10: analyze the single base in heteropolymer

10.1 read the single base in heteropolymer

As described, by two electrodes, all use the spy of 4-sulfydryl benzamide functionalization above in embodiment 4-5Pin and substrate, analyze d(CCACC) oligomer. Observe the characteristic outburst of electric current, the example shows in accompanying drawing 54b(spike of " * " mark is nonspecific, from analyze, gets rid of). Background tunnel current is 10pA, bias voltage+0.5V. As belowShow, low frequency, large-amplitude pulse is designated as C, and high-frequency pulse signal is by a small margin A. Accompanying drawing 54c has shown spike widthThe sliding average (0.25s window, 0.125s ladder) of degree. Value lower than straight line has been identified A base clearly. Accompanying drawing 54d is aobviousShow the sliding average (as defined in the spike to every a pair of vicinity) of pulse frequency, increased at the low frequency region of each endStrong confidence level, is used described confidence level those regions can be assigned as to C base. C base has produced lower than 0.015nA(redLine) insignificant spike number. In accompanying drawing 54e, show the probability that is assigned as A or C. The calculating of these probability is based onThe research of nucleotides, homopolymer and the heteropolymer of this description. This embodiment has clearly illustrated side in complete DNA molecularThe wing is that the single A base of C base can be identified with high confidence level.

10.2 have shown current locus and the d(CCACC of the outburst of nucleotides) long-time track

Accompanying drawing 38 has shown along with probe is with d(CCACC) drift in the tunnel of the generation 10s cycle on the surface that coversThe sampling of noise. Accompanying drawing 39 has shown the typical signal " outburst " from each nucleotides. In accompanying drawing 38, show d(CCACC) typical 10s time locus. Notice the advantage of a-signal. Current spike distributes (illustration) almost completely by " A "Signal is dominated, and the C part (referring to the curve in less frame) in matching is 7% or lower. This has shown that probe cost is moreTime is attached to the A base of minority. In accompanying drawing 39, the nucleotides breaking out for the typical case who has shown data, exists longerTime locus. Each of these examples by the galvanic areas without spike around.

Embodiment 11: with the electrode analysis DNA oligomer of 4-sulfydryl benzamide functionalization

Accompanying drawing 58a, c and e have shown d(A)₅、d（C）₅And d(^mC）₅Representational Tunnel Noise track, corresponding electric currentPeak is distributed in accompanying drawing 58b, d and f and shows. Comparative drawings figs 58b(d(A)₅) and accompanying drawing 56g(dAMP), accompanying drawing 58d(d(C)₅) andAccompanying drawing 57h(dCMP), accompanying drawing 58f(d(^mC）₅) and accompanying drawing 56i(d^mCMP) represented, surprisingly, large during tunnel connectsThe polymer of part has produced the similar signal producing with single core thuja acid in conjunction with event. This discovery shows, (1) is singleBase is read, and (2) are because the space constraint of polymer backbone does not hinder base binding events led signal.

Between nucleotides and oligomer signal, have some (little) difference, these are: (1) peak position, width and phaseIntensity is changed a little. (2) nucleotides produce nearly all signal under 0.5V bias voltage lower than 0.1nA(table 4). PhaseUnder ratio, d(A)₅And d(^mC）₅20% of the resultant signal producing is greater than under this bias voltage that 0.1nA(table 5(below discusses), thisIn accompanying drawing 56G-J, be not significantly, wherein distributing only reaches 0.1nA and is marked and drawed). At d(A)₅And d(C)₅In theseHigh electric current (> 0.1nA) feature is continuous distributed, thus they do not represent that once exceeding the parallel of a base reads (whereinElectric current will distribute on individual molecule value multiple ". But they are relevant to the existence of polymerism structure in tunnel gapNew feature. Spike nonspecific, large amplitude like this marks with an asterisk in accompanying drawing 54b.

I in the oligomer of mixed sequence > characteristic frequency of 0.1nA occurs more much lowerly, shows they and homopolymerIn base stacking relevant. Accompanying drawing 58h has shown d(ACACA) CURRENT DISTRIBUTION, wherein 95% event is lower than 0.1nA. Accompanying drawing58j has shown d(C^mCC^mCC) CURRENT DISTRIBUTION, wherein 99% event is lower than 0.1nA. Real red line is to gather corresponding to the same of componentThe summary of the distribution that thing is measured, calibration aside, an only fitting parameter. This parameter is A/C(rfit=0.48) or mC/C(rfit=0.66) ratio, the rfit of contribution. These values are different from known proportion of composing, and (ACACA is 0.6, C^mCC^mCC is0.4), but be surprising, because the spike rate of independent dCMP is very little, and C seems the sequence of mixingIn oligomer data, very well presented. This shows, by A around C be read more frequently, may be because contain C'sOligomer score from dCMP be attached to better substrate.

Importantly, the oligomer of mixing has produced to a great extent the summation described signal by base independently signal(in accompanying drawing 58h and j, be labeled as some intermediate current reading result of " 1 ", and other a small amount of high electric currents of mark " 2 "Feature, has shown that sequence background has played little effect).

These experiment in, probe drifts about randomly on sample, thus sequence not by certainty " reading ". AlthoughSo, the track that replace of signal between " A sample " and " C sample " (accompanying drawing 58g) and " mC sample " and " C sample " can easily be sent outExisting. The duration (referring to accompanying drawing 57 and embodiment 11) of these signals " outburst " be long (0.14 ± 0.02s in ACACA,0.15 ± 0.02s in CmCCmCC). Similarly outburst is seen in homopolymer (table 5) and nucleotides (table 4).

Table 5: oligomer Tunnel Noise characteristic. Parameter defines in accompanying drawing 57

* the error in the matching of exponential distribution.^§Standard error.

This has produced another unexpected discovery, that is, and combination in interval (ms) between noise spike or solutionThe life-span (very short) of state is compared, and in tunnel gap, the life-span of the compound of combination is (mark of second) grown very much.

The t of embodiment 12: embodiment 11_onAnd t_offDistribution

The current spike that duration is less than 0.1ms is to be changed by slow (10kHZ) response of current-to-voltage converter,And the pulse of exceedance ms duration is subject to the impact of the feedback for maintaining tunnel gap. In accompanying drawing 41, show monomerT_onDistribution, in accompanying drawing 42, shown oligomer. In accompanying drawing 43, show the t of monomer_offDistribution, aobvious in accompanying drawing 44Show oligomer. Solid line is the matching to exponential decay:

Obtain τ on and the τ off value in table 4 and 5, listed.

Accompanying drawing 41 has shown the distribution of the on time of dGMP, dCMP, dAMP and dmCMP. Solid line is that index matching is (from topPortion: Article 1 line is GMP, Article 2 line is CMP, Article 3 line is AMP, Article 4 line is 5meC). Accompanying drawing 42 has shown d（C）₅、d（A）₅And d(mC)₅The distribution of on time. Solid line is that index matching is (from top: Article 1 line is CCCC, Article 2Line is AAA, and Article 3 line is 5mCCC). Compared with monomer, distribution be lower well separate. Accompanying drawing 43 has shownThe distribution of the off time of dGMP, dCMP, dAMP and dmCMP. Solid line be index matching (from top: Article 1 line is CMP,Two-lines is GMP, and Article 3 line is 5meC, and Article 4 line is AMP). Accompanying drawing 44 has shown d(C)₅、d（A）₅And d(mCMP)₅'sThe distribution of off time. Solid line is that (from top: Article 1 line is AAAAA, Article 2 line is 5mCCCC, Article 3 in index matchingLine is CCC). Again, compared with monomer, distribution be lower well separate.

Embodiment 13: the long-life of test compound thing

Power spectrum is used as the unexpected length of 4-sulfydryl benzamide-base-4-sulfydryl benzamide compoundThe independent test in life-span, in the nano-scale gap that described compound is limited in preparing according to embodiment 4-5. In these measurementsIn (accompanying drawing 59a), identification one of molecule is incorporated into AFM probe by polyethylene glycol (PEG) junction of 34nm length, and anotherIndividual at Au(111) form individual layer in substrate. DAMP is used as the target analyte of bridge gap. In the time not there is not dAMP, visitAdhering between pin and substrate is extremely little, presumably because the hydrogen bonding site on benzamide identification molecule by waterMolecule is combination stably. In the situation that there is a small amount of dAMP, observe enclosing characteristic, reduce along with the raising of dAMP concentration(causing that probe and substrate are by dAMP combination). The extension that PEG fastens thing has produced characteristic signal, and it allows multiple combination thingsPart (accompanying drawing 59b(i)) separate with single molecule events (accompanying drawing 59b(ii), thus analyze only single molecular scission event. DoFor the unimolecule bond fission power of the function of hoisting velocity is summarized in accompanying drawing 59c, (solid line is the maximum possible to allos key modelProperty matching), show in accompanying drawing 59d as the key survival probability of the function of bond fission power. Solid line is to same allos key mouldThe matching of type. Under zero-g, they have produced off and have led:

。

Thereby intrinsic (zero-g) time-to-live of this compound is on the order of magnitude of second, instead of millisecond. Also analyzeProduce the distance of the transition state of dissociating, α=0.78nm(and its variation, σ=0.19nm). Thereby conclude, eachBase residence in tunnel connects mark of significant second, and produce the tunnel signal that kHz leads. Thereby, in once breaking out, go outComplete bunch (Burst duration is listed in table 4 and 5) of existing signal can be for characterizing base.

Previous in STM image and in CNT monomolecular reaction on having reported at electronics mark in the impact of transhipmentIn note, be accompanied by the long bonding state life-span of quick fluctuating. The source of this noise is unclear, and just it looks like heightSpend heat sensitively, shown that the little energy of action to causing noise hinders. Analyze the distribution of " on " and " off " time(referring to accompanying drawing 57). In limited time range, the amplifier response by one end and the servo control mechanism of the other end ringBetween seasonable, measure, these distributions are index (as desired to Poisson source), and the 1/e time, (τ on and τ off) existedIn table 4 and table 5, list. Their difference is little, the energy difference calculated Δ G below basis between on and off state:

。

Be created in the value listed in table 4 and 5 (in the unit of heat energy, the k under 300K_BT). These values are all k_BT dividesNumber. Thereby " switching " do not represent the thermal activation (the normal signal source of dual intensity level noise) on significant obstacle. A kind of possibilityExplanation be by the Brownian movement in the combination state of the matrix element sampling of index sensitivity.

" on " and " off " time are so extensively to distribute, thereby they are not very useful for qualification base signal. But the frequency (fS, table 4 and 5) within an outburst is parameter more simply too much. Accompanying drawing 49 and 50 has shown three kindsThe CURRENT DISTRIBUTION of homopolymer and frequency distribution, it is unified being made each area under a curve by standardization. D(mC)₅FrequencyDistribution is bimodulus, to being labeled as f(mCMP on independent mCMP(figure)) observe the many knots that read in " C " frequency rangeReally, and the numeral of very fast speed (approximately 1300Hz). This shows, the binding pattern of mC is shown in polymer environmentChange (conforming to the larger displacement of comparing signal oligonucleotide, polymer signal, accompanying drawing 58f), therefore we select to divide outstandinglyAnalyse the oligomer that contains A and C, particularly the previous d(CCACC analyzing) sequence.

Average current in given outburstAnd frequency, the distribution showing in accompanying drawing 52a-b

(accompanying drawing 52a) and(accompanying drawing 52b)

Determine that base is A or the independent probability of C:

And。

From d(CCACC) CURRENT DISTRIBUTION (illustration in accompanying drawing 38) almost completely by A spike dominate (C in this matchingDistribute part be 7% or still less). This is surprising result, and in sequence, more C has obtained the C spike of smaller amounts.But it is consistent with our hypothesis, in the time that base flank is A, the frequency of C reading result be enhanced (referring to, with dCMPvs.DAMP counting rate is compared, at d(ACACA) in the raising of C reading result). Analysis in conjunction with us to detonator signal, can carry outRationed (this is undertaken by " human eye " in accompanying drawing 58g and i) of composite signal. D(C)₅Do not produce the letter lower than 0.015nANumber, thereby can distribute to clearly A lower than the outburst of the electric current of this level (but higher than noise). For by a larger marginSignal, frequency of utilization and amplitude data. Result is the curve pair showing in accompanying drawing 54e. Make to come in this way sequenced dnaNeed several further development. First, polymer must be dragged tunnel to connect with controlled speed, particularly will read with poly-When thing samsara. Because DNA is too fast and can not read through the nano-pore of functionalization not, base is in the tunnel of functionalization connectsLong remaining time is a kind of advantage. At present, be by uncontrolled mechanical drift from a site to the movement in another siteDrive, it produces unknown power to reading compound. Our power Spectral data can be for obtaining the thick of " pulling " powerSlightly estimate, this realizes given read rate required (it is representative that the off of the measurement of supposition dAMP leads all bases being). Bell equation has provided off under power F and has led and be:

=0.28s^-1And α=0.78nm, 19pN will cause passing through of 10 bases per second. 10 base s^-1Speed read and obtain approximately 30 data spikes (average), the distribution that is enough to produce reasonable confidence level for " C ". 19pNPower can produce by the bias voltage of the only 89mV across nano-pore 17, thereby reading of connecting of 10 bases each tunnel per secondThe rate of getting looks like feasible.

Embodiment 14: the high electric current afterbody in CURRENT DISTRIBUTION

Accompanying drawing 45 has shown the electrode pair d(A of 4-sulfydryl benzamide-functionalization)₅Spike > 0.1nA counting pointCloth. These are total approximately 20%, at dNTP or d(C5) in do not observe. Accompanying drawing 46 has shown d(mC)₅Spike > 0.1The distribution of nA counting. These are total approximately 20%, at dNTP or d(C5) in do not observe.

Embodiment 15: in gold substrate the interactional SPR of nucleosides monophosphate and 4-sulfydryl benzamide estimate andThe combination state life-span in solution

Surface plasma body resonant vibration (SPR) sensor figure is recorded in BI-2000SPR system (BiosensingInstrument, Tempe, AZ) upper, described system equipment has two-pass flow pond, by PAEK (PEEK) pond piece andDimethyl silicone polymer (PDMS) pad composition. Incident light wavelength is 635nm. Before each experiment, with ethanol and two steamingWater cleans flow cell.

By in the coated device (QuorumEmitechCorporation, model K675XD) of sputter at BK7 glass coverThe upper continuously coated 2nm thickness chromium film of slide (VWR#_48366067) and 47nm thickness gold film are manufactured spr sensor chip.For gold substrate, deionized water, straight alcohol clean, and nitrogen dries up, then hydrogen flame annealing before using. The individual layer of benzamide is logicalCross the ethanolic solution that uses continuous passage pattern to inject online 1-sulfydryl benzamide to shape on the golden chip being placed on SPR instrumentBecome. Use is bonded to the molecule of gold surface, and spr signal improves, and finally reaches stable response, shows the maximum coverage of individual layer.The interaction on nucleosides-the 5 '-monophosphate of four kinds of natural generations and benzamide surface is used single-channel mode on SPR instrumentMeasure. The sample solution of injecting by introduction valve flows by a passage, and PBS buffer solution (pH7.4,10mM phosphoric acidSalt and 150mMNaCl) flow through another. Measure in PBS buffer solution under the nucleosides monophosphate concentration of 1mM with 60 μ Lmin^-1Flowing velocity carry out.

In the software that data analysis provides sale business, carry out. The simple 1:1 interaction model of all data set matchings.

The uncertain K of data_off, but very large K_DValue (several mM) means that off leads fast. For example, suppose K_on=10⁶M^-1s^-1(little) value, mMKD produces K_off=K_DK_on=103 or in conjunction with the ms time scale in state life-span.

Accompanying drawing 47 has shown and benzamide surface (R: the 2-deoxyribosyl 5-sodium ascorbyl phosphate that does not contain DNA base) phaseThe spr sensor figure of nucleosides-the 5 '-monophosphate (A, C, G, T, R) of mutual effect. Red line is the curve of matching, is modeled to describe1:1 binding events. Table 6 has shown speed constant and the dissociation constant analyzed derived from 1:1 binding kinetics.

Table 6

。

Embodiment 16: the frequency of bond fission reading result in power spectroscopy

In the case of exist independent buffer solution test interact (accompanying drawing 48a---shown and pulled it 1024 timesIn the detected event of adhering to only) afterwards, 1 μ MdAPM adds the liquid with the electrode configuration of 4-sulfydryl benzamide functionalization toIn body pond, then gather force curve as the function of substrate wash number, tip is 0.1mMPB. Excessive along with removingDAPM data have shown raising originally, are subsequently to follow the reduction (accompanying drawing 48b-e) that continues cleaning. Particularly, accompanying drawing 48 is aobviousShown that the control curve that (a) gathers while not there is not dAPM shows the event of adhering between benzamide molecule hardly, supposition isBecause they are broken by water resistance. Add dAMP cause many events of adhering to, its along with excessive dAMP washed out system and improve (b,C), reduce (d, e) along with cleaning continuation.

Embodiment 17: noise model

The object of this embodiment is the possible source of resolving for the identification of the signal of base. As shown in embodiment 13, in tunnel connects, the original life of the compound of combination is long---on the order of magnitude in second. If thereby electrode orProbe is by translation, and base is combination all the time in the time of the electrode of its contiguous functionalization generally, thus the even several bases of reading per second. ?What does is the source of the current spike repeating in ms time scale? dividing of " on " providing in embodiment 12 and " off " timeCloth can be for calculating the energy difference between " on " and " off " state. This lists with Δ G in table 5. This is heat energy under 300KMark (unit is kT, under 300K). Thereby spike can not be the result that molecule jumps between unique thermal steady state.At this, we have studied " spike in the time sampling in index mode (, by the tunnel-effect of the index sensitivity of adjusting the distance)Ground " there is the possibility of continuous Brownian movement. Notice, this model-composing electric current logarithm exponential distribution selectBasis (the equation S1 in embodiment 8). With Gaussian(, heat) the random walking machine of the 1-D simulation Blang fortune that drives of noiseMoving. Displacement is carried out by indexation the impact that the tunnel current of analog position is read. Use following MatLab program:

forx=2:10000

z=randn（1）；

y（x）=correlation*y（x-1）+0.1*z；

end

a=exp（beta*y）；

plotyy（t,y,t,a）

Variable " correlation " has been described position in a step to be had and how much in next step, retains. ?In accompanying drawing 49-51, the various values of parameter " correlation " are shown to curve. To obtain similar observing to make an uproar near 1 valueThe noise spike of sound is required. Intensity distribution well with the logarithm Gaussian matching (reference, equation S1) of electric current, spike itBetween the time interval distribute exponentially. Accompanying drawing 49-51 has shown the displacement of the simulation of three kinds of values of correlationC(top reading result) and electric current (bottom reading result) reduced time-ladder.

Accompanying drawing 18: probability calculation

Accompanying drawing 52 has shown in the equipment of electrode with 4-sulfydryl benzamide functionalization, has obtained from homopolymerThe standardized distribution of signal. Accompanying drawing 52A is fit to standardized CURRENT DISTRIBUTION (from left to right: first peak=mC, secondPeak=A, the 3rd peak=C). Accompanying drawing 52B shown at the peak frequency of signal outburst Plays (fS---referring to accompanying drawing 57),With (at the line of about 0.2=A place beginning, at the line of about 0.65=C place beginning, opening at about 0.4=mC place of multinomial measurement and matchingThe line beginning). To the matching distributing be used to specify specific noise outburst from the probability of A or C (if average current and frequency existAbove or below crosspoint, be labeled as " IAC " and " fAC "). The CURRENT DISTRIBUTION of C and mC is the (intersection=" I separating_mC"), stillFrequency distribution is overlapping.

Value:

With

Take from accompanying drawing 52a. Owing to there is no the current spike lower than 0.015nA for C, mean intensity is less thanThe outburst of 0.015nA can be distributed to A reading result. For intensity > outburst of 0.015nA, we have used value:

With

It takes from standardized distribution accompanying drawing 52b, the probability from following calculating A reading result:

Probability from following calculating C reading result:

。

Embodiment 19: imidazoles-2-carbamyl synthetic

It is required that imidazoles-2-carbamyl is attached to electrode by the alkyl of short ω-functionalization. Due to various 4(5)-alkaneThe imidazoles of base reported in the literature, or commercially available, developed on imidazole ring and synthesized by amidatioonThe conventional method of imidazoles-2-carbamyl. As described in following scheme, 4(5)-(2-sulphur ethyl) imidazoles-2-carbamyl (5a) and4(5)-(2-aminoethyl) imidazoles-2-carbamyl (5b) is respectively by amidatioon 4(5)-(2-(benzylthio) ethyl) imidazoles (1a)With N-[2-(4-imidazole radicals) ethyl] phthalimide (1b) synthesizes. Sulphur alkohol and amine is as anchor group, for molecule attached is arrivedMetal and/or carbon electrode. With same method, 4(5)-(tert-butyl dimethyl siloxane methyl) imidazoles-2-carbamyl (5c)From 4(5)-(uncle-Ding dimethyl siloxane methyl) imidazoles (1c) is synthetic, and its NMR being used in organic solvent studies (under seeingLiterary composition).

Two kinds of approach of synthetic these imidazoles-2-carbamyls are explored. Formic acid esters can be used in the 2-position of imidazoles¹⁵Or cyano groupGroup,¹⁶Replace, the two is all easily converted into acid amides. What find is cyano group approach for we provide best result. FirstFirst, by react with cylite with good productive rate by compound 1a, 1c and 1b be converted into the product that 1H nitrogen protects (2a, 2b,2c). NMR confirmed them each be the mixture of two kinds of isomers. By using 1-cyanogen-4-(dimethylamino) pyrrolePyridine bromide (CAP) is processed them, cyano group is imported to the 2-position of the imidazole ring of 2a, 2b and 2c. By at dimethylIn formamide (DMF), mixing a considerable amount of cyanogen bromides and 4-(dimethylamino at 0 DEG C) pyridine produces CPA in situ. WithBest output produces the CAP of 2.5 times. By hydrolysis in sulfuric acid (by volume 20%) and trifluoroacetic acid (by volume 18%), withStill can productive rate the cyano group of 3a, 3b and 3c is converted into acid amides (4a, 4b, 4c). We have tested and have had hydrogen peroxideSituation under alkali condition, but fail to provide the product of expectation. The end product of 5a, 5b and 5c by using sodium in liquefied ammoniaRemoving blocking group obtains. It should be noted that tert-butyl dimethyl siloxane methyl group is steady under deprotection conditionFixed. The compound 5c expecting separates with good productive rate.

Embodiment 20: with the electrode analysis DNA of imidazoles-2-carbamyl functionalization

Prepare electrode and use imidazoles-2-carbamyl functionalization. Configure fixing tunnel gap, with the base of 6pA, 0.5VThreaded list road condition analysis deoxidation-nucleotides. Control group does not almost have display. Accompanying drawing 62A and B have shown^mC, A, T, C and GCURRENT DISTRIBUTION. Be apparent that, the electric current mark of every kind of nucleotides is unique, thereby has represented imidazoles-2-carbamyl workFor the validity of reagent.

Embodiment 21: with the electrode analysis DNA oligomer of imidazoles-2-carbamyl functionalization

The electrode of embodiment 20 is used to analyze several same aggressiveness and different aggressiveness. An electrode is configured to by constant clearanceTranslation on electrode surface, as explanation in accompanying drawing 63. The typical rate of translation is about 8.6nm/s, and it has been simulated and has been pulled through exampleAs the DNA of nano-pore.

Accompanying drawing 64 has shown d(CCCCC) (accompanying drawing 64A) and d(AAAAA) the exemplary CURRENT DISTRIBUTION of (accompanying drawing 64B). WithThe result reading continuously of polymers is summarized (AAAAA---accompanying drawing 65 in accompanying drawing 65-68; CCCCC---accompanying drawing 66; d（^mC）₅---accompanying drawing 67; And d(CCCCC) (accompanying drawing 68)), mark and draw electric current (nA) by the time (s). Reading result has been shown with poly-The consistent signal of thing.

The result reading continuously of heteropolymer is summarized (ACACA---accompanying drawing 70 in accompanying drawing 70-75; CCACC---accompanying drawing71；C^mCC^mCC---accompanying drawing 72; D(ACACA)---accompanying drawing 73; D(C^mCC^mCC)---accompanying drawing 74), and d(GTCGTCGTC)---accompanying drawing 75), mark and draw electric current (nA) by the time (s). Reading result has been shown the consistent letter of heteropolymerNumber.

Thereby, above-mentioned testing authentication imidazoles-2-carbamyl be to analyze the potent agent of oligomer.

Synthesizing of embodiment 22:4-carbamyl phenyl dithiocar-bamate

To DMF(1mL) in the solution of 4-aminobenzamide (2mmol, 272mg), as follows at 0 DEG C continuouslyAdd in NaH(mineral oil 60%, 1.2eq, 9.6mg) and CS2(1.5eq., 181.3uL).

At 0 DEG C, after 30 minutes, reactant mixture is heated to room temperature, stirs 84 hours, is then heated to 60 DEG C and stir4 hours. After cool to room temperature, ether for reactant mixture (20mL) dilution, filters, and washs to obtain yellowish powder with ether183mg(productive rate 39%).

It being understood that various above-disclosed contents and other features and function, or its selectable content, Ke YiliWant to be combined into many other different systems or application. And, various current that do not expect or do not anticipate at thisOptional content, amendment, variation or improvement, can be made subsequently by those skilled in the art, also expect by following rightRequirement is contained.

<110>ArizonaBoardofRegents

Lindsay,Stuart

Chang,Shuai

He,Jin

Zhang,Peiming

Huang,Shuo

<120>for the controlled tunnel gap equipment of the polymer that checks order

<130>2912941-023977

<150>U.S.61/300,678

<151>2010-02-02

<150>U.S.61/378,838

<151>2010-08-31

<160>12

<170>PatentInversion3.5

<210>1

<211>5

<212>DNA

<213>artificial sequence

<220>

<223>oligomer

<400>1

ccacc5

<210>2

<211>5

<212>DNA

<213>artificial sequence

<220>

<223>oligomer

<400>2

acaca5

<210>3

<211>5

<212>DNA

<213>artificial sequence

<220>

<223>oligomer

<220>

<221>misc_feature

<222>(1)..(5)

<223>n is 5-methyl-deoxycytidine

<400>3

cncnc5

<210>4

<211>5

<212>DNA

<213>artificial sequence

<220>

<223>oligomer

<400>4

aaaaa5

<210>5

<211>5

<212>DNA

<213>artificial sequence

<220>

<223>oligomer

<400>5

ccccc5

<210>6

<211>5

<212>DNA

<213>artificial sequence

<220>

<223>oligomer

<220>

<221>misc_feature

<222>(1)..(5)

<223>n is 5-methyl-deoxycytidine

<400>6

nnnnn5

<210>7

<211>9

<212>DNA

<213>artificial sequence

<220>

<223>oligomer

<400>7

gtcgtcgtc9

<210>8

<211>4

<212>DNA

<213>artificial sequence

<220>

<223>oligomer

<400>8

cccc4

<210>9

<211>3

<212>DNA

<213>artificial sequence

<220>

<223>oligomer

<400>9

aaa3

<210>10

<211>3

<212>DNA

<213>artificial sequence

<220>

<223>oligomer

<220>

<221>misc_feature

<222>(1)..(3)

<223>n is 5-methyl-deoxycytidine

<400>10

ncc3

<210>11

<211>4

<212>DNA

<213>artificial sequence

<220>

<223>oligomer

<220>

<221>misc_feature

<222>(1)..(4)

<223>n is 5-methyl-deoxycytidine

<400>11

nccc4

<210>12

<211>3

<212>DNA

<213>artificial sequence

<220>

<223>oligomer

<400>12

ccc3

Claims (19)

1. for an equipment for analyzing polymers, described equipment comprises

(a) the first and second electrodes, they form the tunnel gap that described polymer can pass, wherein said tunnel gap toolThere is adjustable width; With

(b) be attached to the first reagent of described the first electrode and be attached to the second reagent of described the second electrode, wherein said theOne and second reagent can form instantaneous key with the unit of polymer separately,

Wherein in the time that forming, instantaneous key produces detectable signal,

Wherein said the first and second reagent independently selected from by mercaptobenzoic acid, 4-sulfydryl phenylurea, imidazoles-2-carbonyl compound andThe group that 4-carbamyl phenyl dithiocar-bamate forms.

2. the equipment of claim 1, wherein, the width of described the first and second reagent and described tunnel gap is configured in instituteWhen the described unit of stating the first and second reagent and polymer forms instantaneous key, produce specific to every kind of monomer of polymerDetectable signal.

3. the equipment of claim 1, the wherein said first and/or second electrode comprises gold, carbon, platinum, Graphene or titanium nitride.

4. the equipment of claim 1, the detectable signal of wherein said generation is the only list by polymer in tunnel gapThe combination of unit causes.

5. the equipment of claim 1, the width of wherein said tunnel gap is from 1 to 4nm capable of regulating.

6. the equipment of claim 1, wherein said the first and second reagent are identical.

7. the equipment of claim 1, wherein said instantaneous key is hydrogen bond.

8. the equipment of claim 1, at least one hydrogen bond donor in wherein said the first and second regent pack property of water-bearing solutionWith at least one hydrogen bond acceptor.

9. the equipment of claim 1, wherein said polymer is DNA or RNA, described unit is nucleotides.

10. the equipment of claim 9, wherein said unit select free A, C, T, G and^mThe group that C forms.

The equipment of 11. claims 1, wherein said polymer is protein, described unit is amino acid.

The equipment of 12. claims 1, further comprises

C) first fluid holder and second fluid holder, at least one nanometer that it can be flowed through by described polymerSeparate in hole.

The equipment of 13. claims 12, further comprises the first drive electrode and the second drive electrode, comes at described first fluidBetween holder and described second fluid holder, apply electrophoresis bias voltage.

The equipment of 14. claims 12, further comprises the top contact electrode that is positioned at described nano-pore top, wherein said receivingRice hole is to conduct electricity in electricity.

The equipment of 15. claims 1, wherein said the first and second electrodes are CNTs.

The method of 16. 1 kinds of analyzing polymers or polymer unit, described method comprises:

A) between the unit of polymer and the first electrode by the first reagent functionalization, and the unit of described polymer and useBetween the second electrode of the second reagent functionalization, form instantaneous key, it is passable that described the first and second electrodes form described polymerThe tunnel gap of passing, wherein said tunnel gap has adjustable width; And

B) in the time that forming, instantaneous key detects detectable signal,

Wherein said the first and second reagent are identical, and select free mercaptobenzoic acid, 4-sulfydryl phenylurea, imidazoles-2-carbonylThe group that compound and 4-carbamyl phenyl dithiocar-bamate form.

The method of 17. claims 16, wherein said polymer is DNA or RNA, described polymer unit is nucleotides.

The method of 18. 1 kinds of polymer that check order, described method comprises

The unit of a) allowing described polymer flows at the first electrode with the first reagent functionalization and by the second reagent functionalizationThe second electrode between the tunnel gap that forms, wherein said tunnel gap has adjustable width;

B) between the unit of described polymer and described the first and second reagent, form instantaneous key;

C) in the time that forming, instantaneous key detects detectable signal; And

D) to the each continuous unit repeating step of polymer a)-c),

Wherein said the first and second reagent independently selected from by mercaptobenzoic acid, 4-sulfydryl phenylurea, imidazoles-2-carbonyl compound andThe group that 4-carbamyl phenyl dithiocar-bamate forms.

The equipment of 19. claims 1, wherein said instantaneous key is in the aromatic group and analyte in reader moleculePi-between aromatic group is stacking.